Analogues of human thyrocalcitonin

ABSTRACT

1. PEPTIDES OF THE FORMULA I H-CYS-GLY-ASN-LEU-SER-THR-CYS-MET-LEU-GLY-THR-TYR-THR-GLNASP-PHE-ASN-LYS-PHE-HIS-THR-PHE-PRO-GLN-THR-ALA-ILEGLY-VAL-GLY-ALA-PRO-NH2, WHERE THE CYS RESIDUES ARE JOINED, IN WHICH THE AMINOACID IN POSITION 8 IS REPLACED BY VALINE, NORVALINE, LEUCINE, ISOLEUCINE, NORLEUCINE OR A-AMINOBYTYRIC ACID, AND THOSE WHICH CONTAIN GLYCINE IN POSITION 12 AND THOSE WHICH CONTAIN METHIONINE IN POSITION 8 AND GLYCINE IN POSITIONS 12 AND 18, OR AT LEAST IN POSITION 12, THEIR ANTIPARALLEL DIMER AND NA-ACYL DERIVATIVES WHEREIN ACRYL IS LOWER MONOBASIC OR DIBASIC ALKANOYL, LOWER ALKYLOXYCARBONYL, BENZYLOXYCARBONYL, L-PYROGLUTAMYL, CARBAMOYL, N-LOWER ALKYLCARBAMOYL, N-PHENYLCARBAMOYL, OR N-PHENYLTHIOCARBAMOYL, OR DESAMINO1-DERIVATIVES OF THE MONOMERIC OR DIMERIC PEPTIDES, AS WELL AS THERAPEUTICALLY ACCEPTABLE ACID ADDITIONS SALTS AND COMPLEXES WITH A MEMBER SELECTED FROM THE GROUP CONSISTING OF ZINC PHOSPHATE, ZINC PYROPHOSPHATE, ZINC HYDROXIDE, GELATINE, POLYPHLORETIN PHOSPHATE AND POLYGLUTAMIC ACID, OF THE SAID MONOMERIC OR DIMERIC PEPTIDES WITH THE PROVISO THAT ALL AMINOACID RESIDURS OTHER THAN GLYCINE ARE TO THE L-CONFIGURATION.

United States Patent 3,849,388 ANALOGUES OF HUMAN THYROCALCITONIN Werner Rittel, Basel, Max Brugger, Birsfelden, Bruno Kamber, Basel, Bernhard Riniker, Frenkendorf, and Peter Sieber, Reinach, Basel-Land, Switzerland, and Hendrik Marie Greven, Heesch, Netherlands, assignors to Ciba-Geigy Corporation, Ardsley, N.Y.

No Drawing. Continuation of abandoned application Ser. No. 864,544, Oct. 7, 1969. This application Sept. 28, 1972, Ser. No. 293,271

Claims priority, application Switzerland Oct. 15, 1968,

15,401/68; Nov. 20, 1968, 17,286/68 Int. Cl. A61]; 27/00; C07c 103/52 U.S. Cl. 260-1125 14 Claims ABSTRACT OF THE DISCLOSURE The new peptides of the formula I 13 14 15 1e 17 1s 19 20 21 22 23 24 Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln- 25 26 27 2s 29 31 32 Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NHi (I) in which at least one and at most 16 of the aminoacids in the positions 2, 8, 10, 11, 12, 13, 14, 16, 18, 21, 23, 24, 25, 26, 27, 29, 30 and 31 are replaced by other natural a-amino-acids or their homologues are useful as hypocalcaemic agents and are prepared by splitting off groups protecting at least one amino group or carboxyl group or oxidizing the corresponding mercapto compounds to the disulfides or condensing together adequate peptides.

CROSS-REFERENCE TO RELATED APPLICATION This is a continuation of application Ser. No. 864,544, filed Oct. 7, 1969 (now abandoned).

The present invention provides new hypocalcaemically active peptides of the formula Thr-Gin-Asp-Phe-Asn-Lys-Phe His-Thr-Phe-Pro-Gln- Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH;

in which at least one and at most 16 of the aminoacids in the positions 2, 8, 10, 11, 12, 13, 14, 16, 18, 21, 23, 24, 25, 26, 27, 29, 30 and 31 are replaced by other natural u-amino-acids or their homologues, especially by the aminoacids present in the corresponding positions of porcine thyrocalcitonine, their dimers, especially those in which 2 identical peptide sequences (l-32 and 1'32') are linked in an antiparallel arrangement via the cysteine residues 1,7 and 7,1 through a disulphide bond, and derivatives of the monomeric or dimeric peptides as well as acid addition salts and complexes of the said monomeric and dimeric peptides and their derivatives, and processes for the manufacture of these compounds.

Of special value are those peptides of the formula I, in which the aminoacid in position 8, methionine, is replaced by valine, norvaline, leucine, isoleucine, norleucine or a-aminobutyric acid and those which contain glycine in position 12 and/ or 18, their dimers, derivatives and acid addition salts and complexes.

Derivatives of said compounds are primarily those in which the a-amino group is acylated, as well as corresponding desamino -peptides.

Acyl groups suitable for the acylation of the amino groups, especially for the acylation of the N-amino group, are the residues of carboxylic acids such as aliphatic, aromatic, araliphatic, heterocyclic and heterocyclyl-aliphatic 3,849,388 Patented Nov. 19, 1974 icecarboxylic acids, especially of lower monoor dibasic alkane or alkene acids such as formic, acetic, propionic, a butyric, acrylic, succinic acid, of alicyclic carboxylic acids such as cycloalkylcarboxylic acids, of monoor dibasic monocyclic aromatic carboxylic acids such as unsubstituted or substituted benzoic acid or phthalic acid, of unsubstituted and aryl-substituted aryl-lower alkyl or -alkenyl-carboxylic acids such as phenylacetic acid, of unsubstituted or substituted monoor dibasic 5- or 6-membered heterocyclic acids with nitrogen, sulphur and/or oxygen as hetero atoms, such as pyridinecarboxylic acids, thiophenecarboxylic acids, or of heterocyclyl-lower alkanoic acids such as pyridylacetic acid, imidazolylacetic acid in which the rings are substituted, for example, by halogen atoms, nitro groups, lower alkyl or lower alkoxy groups or lower carbalkoxy groups. :Further specially suitable acyl groups are the acyl residues of aminoacids, especially a-aminoacids, for example the pyroglutamyl residue; furthermore acyl radicals derived from carbonic or thiocarbonic acid or from their esters or amides, for example lower alkyloxycarbonyl groups such as ethoxycarbonyl, tertiary butoxycarbonyl; also benzyloxycarbonyl, carbamoyl or thiocarbamoyl which may be unsubstituted or substituted as indicated above, also N-substituted can bamoyl and thiocarbamoyl, for example N-lower alkylcarbamoyl, N-phenyl-carbamoyl and N-phenylthiocarbamoyl.

The acid addition salts are primarily salts of therapeutically acceptable acids such as hydrochloric, acetic, sulphuric, phosphoric acid or of sulphonic acids such as lower alkanesulphonic acids, benzenesulphonic or toluenesulphonic acid.

The complexes are compounds, whose structure has not yet been clarified, which are formed when certain inorganic or organic substances are added to long-chain peptides and prolong their active life. Such substances have been described for instance for ACTH and other adrenocorticotropically active peptides. There may be mentioned, for example, inorganic compounds derived from metals such as calcium, magnesium, aluminium, cobalt or especially from zinc, especially sparingly soluble salts such as the phosphates, pyrophosphates and polyphosphates as Well as hydroxides of these metals; furthermore alkali metal polyphosphates, for example Calgon N, Calgon 322, Calgon 188 or Polyron B-l2. Organic substances that prolong the activity are, for example, nonanitgenic gelatin, for example polyoxygelatin, polyvinylpyrrolidone and carboxymethylcellulose, also sulphonic or phosphoric acid esters of alginic acid, dextran, polyphenols and polyalcohols, in the first place polyphloretine phosphate and phytic acid, also polymers and copolymers of gminoacids, for example protamine and polyglutamic aci The new compounds, especially those which in 8-position contain the aforementioned exchange amino-acids, have a hypocalcaemic action. They lower the plasma-calcium and -phosphate level of the blood of mammals, as was shown by experiments on Wistar rats.

The compounds also lower the plasma-calcium level of human blood on intravenous administration of 0.01 to 5 mg. in 0.1 molar acetate buffer of pH=4.6 and can therefore be used for treating hypercalcaemia.

The present process for the manufacture of the new monomeric or dimeric peptides, their derivatives, their acid addition salts and complexes is characterized in that 16 17 18 19 20 21 22 23 24 25 26 2728 29 30 31 Phe-Asn-Lys-Phe-His-Thr-Phe-ProGln-Thr-Ala-Il -Gly-Val-G1y-Ala- 32 Pro-NH;

in which at least one and at most 16 of the aminoacids in positions 2, 8, 10, 11, 12, 13, 14, 16, 18, 21, 23, 24, 25, 26, 27, 29, 30 and 31 are replaced by other natural aaminoacids or their homologues or corresponding dimers or derivatives of these peptides, in which compounds at least one amino group or a carboxyl group is protected by an eliminable protective group, the protective group(s) is/ are eliminated; or

(2) a compound of the formula II 1 2 3 4 56 7 8 9 101112131415 H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp- 16 17 18 19 20 21 22 23 24 25 26 2728 29 30 31 Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala- Pro-NH:

in which at least one and at most 16 of the aminoacids in positions 2, 8, 10, 11, 13, 14, 16, 18, 21, 23, 24, 25, 26, 27, 29, 30 and 31 are replaced by other natural oraminoacids, or their homologs, or an aforementioned derivative thereof in which compounds the mercapto groups are free or protected by the trityl group, is oxidized to form a disulfide, or

(3) a compound of the formula III or IV S---- (EH2 R-( JH-CO-Y-Asn-Ley-Ser-Thr-Cys-OH or (III) s (EH, R-C H-CO-Y-Asn-Leu-Ser-Thr-Cys-A in which Y represents glycyl or L-seryl, A stands for 1 to 21 of the aminoacid residues following upon cysteine which may contain a protected side-chain amino group and which may have been exchanged in the manner indicated, and R represents hydrogen or an acylated amino group, is condensed with the remaining C-terminal sequence of the peptide with possibly protected side chain amino group (up to the C-terminal aminoacid L-prolineamide) by a method conventionally used in the peptide synthesis, and, if desired, the resulting monomeric compound is converted into its dimers or the free monomeric or dimeric peptides are converted into their derivatives and/ or acid addition salts or complexes.

Protective groups used with special advantage in the manufacture of the starting materials for variant (1) of the present process, and also for all intermediates required in the 3 process variants, are those known from the synthesis of long-chain peptides as well as a few new protective groups that are easy to eliminate, for example by hydrolysis, reduction, aminolysis or hydrazinolysis.

Thus, for example, there are used as protective groups for amino groups acyl or aralkyl groups such as formyl, trifluoracetyl, phthaloyl, benzenesulphonyl, p-toluenesulphonyl, o-nitrophenylsulphenyl, 2,4-dinitrophenylsulphenyl groups (these sulphenyl groups can alternatively be eliminated by treatment with nucleophilic reagents, for example sulphites, thiosulphates; see Kamber and Rittel, Helv. Chim. Acta 51 (1968) 2061), benzyl groups which may be substituted, for example by lower alkoxy, especially or p-methoxy groups, or diphenylmethyl or triphenylmethyl groups, or groups derived from carbonic acid, such as arylmethyloxycarbonyl groups whose aromatic rings may be substituted for instance by halogen atoms such as chlorine or bromine, nitro groups, lower alkyl or lower alkoxy groups or chromogenic groups, for example azo groups, in which the methylene group may be substituted by a further aryl residue and/or one or if desired two lower alkyls, such as benzyl, benzhydryl or 2 phenyl isopropyloxycarbonyl groups, for example carbobenzoxy, p-bromoor p-chloro-carbobenzoxy, p-

nitrocarbobenzoxy or p-methoxy, carbobenzoxy, p-phenylazo-benzyloxycarbonyl and p (p methoxyphenylazo)- benzyloxycarbonyl, 2 tolyl isopropoxycarbonyl and especially 2-(p-biphenylyl)-isopropoxycarbonyl [cf. Kamber and Rittel, Helv. Chim. Acta 51 1968) 2061] also aliphatic oxycarbonyl groups such as adamantyloxycarbonyl, cyclopentyloxycarbonyl, trichloro-ethoxycarbonyl, tertiary amyloxycarbonyl or in the first place tertiary butyloxycarbonyl.

The amino groups can also be protected by formation of enamines obtained by reacting the amino group with a 1,3-diketone, for example benzoylacetone, acetylacetone or dimedone.

Carboxyl groups are protected, for example, by amide or hydrazide formation or by esterification. The amide and hydrazide groups may be substituted, the amide group for instance by the 3,4 dimethoxybenzyl or bis-(p-methoxyphenyl)-methyl group, the hydrazide group for instance by the carbobenzoxy group, the trichlorethyloxycarbonyl group, the trifluoracetyl group, the trityl group, the tertiary butyloxycarbonyl group or the 2-(p-biphenylyl-isopropoxycarbonyl group. For the esterification there may be used, for example, lower, possibly substituted, alkanols such as methanol, ethanol, cyanomethyl alcohol, benzoylmethyl alcohol or especially tertiary butanol, furthermore aralkanols such as aryl-lower alkanols, for example benzyl or benzhydryl alcohols which may be substituted by lower alkyl or lower alkoxy groups or by halogen atoms, such as p-nitrobenzyl alcohol, p-methoxybenzyl alcohol or 2,4,6 trimethylbenzyl alcohol, phenols and thiophenols which may be substituted by electron-attracting substituents, such as thiophenol, thiocresol, p-nitrothiophenol, 2,4,5- and 2,4,6 trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, p-cyanophenol or p-methanesulphonylphenol, also, for example, N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxypiperidine and 8-hydroxyquinoline.

The hydroxyl groups of the serine, threonine and tyrosine residues can be protected, for example, by esterification or etherification. Acyl residues suitable for the esterification are, for example, lower alkanoyl residues such as acetyl, aroyl residues such as benzoyl and in the first place residues derived from carbonic acid such as benzoyloxycarbony] or ethyloxycarbonyl. Groups suitable for the etherification are, for example, benzyl, tetrahydropyranyl or tertiary butyl residues. Furthermore, the hydroxyl groups can be protected by the 2,2,2-trifluoro-ltertiary butoxycarbonylamino or -1-benzyloxycarbonylaminoethyl groups (Weygand) described in Ber. [1967], pages 3838-3849. However, the hydroxyl groups need not necessary be protected.

The mercapto groups of the cysteine residues are protected for instance by acylation or alkylation. The acylation may be carried out, for example, with the acetyl or benzoyl residue, the ethylcarbamyl residue of the (possibly substituted) carbobenzoxy residue. The alkylation may be carried out, for example, with the tertiary butylor benzylthiomethyl residue or a (possibly substituted) arylmethyl group such as benzyl, p-nitrobenzyl, diphenylmethyl, dimethoxybenzhydryl or trityl, also phenylcyclohexyl, thienyl-(2)-cyclohexyl; cf. Ber. 10] [1968], page 681. The imino group of histidine need not necessarily be protected, though it may be advantageous to protect it, for example by benzyl, trityl, carbobenzoxy, adamantyloxycarbonyl or the above-mentioned Weygands groups.

In varient (l) of the present process it is advantageous to protect the carboxyl group of the side-chain with the tertiary butyl ester group for protecting the amino group of the side-chain the tertiary butyloxycarbonyl group, for the hydroxyl groups of the serine, threonine and tyrosine residues (if they are to be protected at all) the tertiary butyl other group and, if desired, for protecting the imino group of histidiue the 2,2,2-trifluor0-1- tertiary butyloxycarbonylaminoethyl group. If desired, all those protective groups can be eliminated in a single stage by acid hydrolysis, for example with trifiuoroacetic or hydrochloric acid. In synthesizing the starting materials.

used in variant (1), that is the protected dotriaconta peptides with the use of protective groups eliminable with trifluoracetic or hydrochloric acid, the mercapto groups are preferably protected by benzylation or tritylation. The S-trityl groups can be eliminated from the protected peptide in an organic solution selectively'(while retaining the groups eliminable with trifiuoroacetic acid) with mercuric acetate and hydrogen sulphide. The S-benzyl groups can be eliminated from the protected peptide selectively with sodium in liquid ammonia. In either case the peptide containing free mercapto groups is obtained which can then be oxidized to the protected disulphide, for example with iodine in glacial acetic acid, or with diiodoethane in an organic solvent or with atmospheric oxygen in liquid ammonia. It is specially advantageous to protect the mercapto groups by trityl groups which are then eliminated from the protected peptide with iodine in methanol with simultaneous formation of the disulphide bridge; cf. Kamber and Rittel, Helv. Chim. Acta 51 (1968) 2061. The disulphide ring can be formed at a stage of a partial sequence containing the two cysteine residues, for example on the decapeptide 1-10, or at the stage of the dotriacontapeptide.

In variant (2) of the present process the open-chain peptide used as starting material is preferably likewise prepared with the protective groups indicated for variant (1). The S-trityl groups can be eliminated with trifiuoracetic acid, and the free open-chain peptide can be oxidized in known manner with potassium ferricyanide in an aqueous solution or with iodine or with air in liquid ammona. Alternatively, the trityl groups can be eliminated as described above with iodine and methanol with simultaneous formation of the disulphide.

In the manufacture of N-acyl derivatives the acyl group may serve as amino protective group.

The resulting monomeric peptides can subsequently be converted in known manner into their dimers and vice versa and/ or the monomeric or dimeric peptides into their derivatives, acid addition salts and/or complexes. The subsequent conversions can be performed in a suitable order, either singly or in combination.

Resulting monomeric may be converted into dimeric compounds, for example by treatment with mercapto 60mpounds in a neutral or weakly acidic medium, for example by treatment with cysteine hydrochloride. The dimeric compounds can be converted into the monomeric compounds under basic conditions, for example with dilute ammonia.

For the manufacture of acyl derivatives the free peptide can be N-acylated in the usual manner, for example by reaction with a mixed anhydride or acid azide containing the desired acyl residue, or in the first place with an activated ester such as phenyl or substituted phenyl ester. If desired, the acylation can be carried out selectively so that only the oc-al'IliIlO group is acylated. I

Acid addition salts are formed in known manner.

Complexes are likewise formed by known or equivalent methods.

Complexes with inorganic substances, such as sparingly soluble metal compounds, for example aluminium or zinc compounds, are preferably manufactured in a similar manner as known for ACTH, for example by reaction with a soluble salt of the metal concerned, for example zinc chloride or sulphate, and precipitation with an alkali metal phosphate and/or hydroxide. Complexes with organic compounds, such as polyoxygelation, carboxymethylcellulose, polyvinylpyrrolidone, polyphloretine phosphate, polyglutamic acid or the like are prepared by mixing these substances with the peptide in an aqueous solution. In an identical manner insoluble compounds with alkali metal polyphosphates can be prepared.

The invention includes also any variant of the present process in which an intermediate obtained at any stage thereof is used as starting material and any remaining step(s) is/are carried out or the process is discontinued at any stage thereof and/or in which a starting material is formed in situ and/or is used in form of a salt thereof.

The peptides used as starting materials are obtained by linking the aminoacids-if required or desired with the use of readily eliminable protective groups-in the indcated order singly or in form of preformed small peptide fragments, and possibly at a suitable stage the disulphide bridge is formed. It is advantageous to employ the suitable linking methods described in the literature for the manufacture of long-chain peptides, taking into consideration the disulphide bridge.

Accordingly, the aminoacid and/or peptide fragments are linked so, for example, that an aminoacid or peptide containing a protected u-amino group and an activated terminal carboxyl group is reacted with an aminoacid or a peptide containing a free a-amino group and a free or protected (for instance esterified or amidated) terminal carboxyl group, or an aminoacid or a peptide containing an activated a-amino group and a protected terminal carboxyl group is reacted with an aminoacid or a peptide containing a free terminal carboxyl group and a protected a-amino group. The carboxyl group can be activated for instance by conversion into an acid azide, anhydride or imidazolide or into an activated ester such as cyanoethyl ester, thiophenylester, p-nitrothiophenyl ester, thiocresyl, p methanesulphonylphenyl, p nitrophenyl, 2,4-dinitrophenyl, 2,4,5- or 2,4,6-trichlorophenyl, pentachlorophenyl, N hydroxysuccinimide, N hydroxyphthalimide, 8-hydroxyquinoline, N-hydroxypiperidine ester, or by reaction with a carbodiimide (optionally with addition of N- hydroxysuccinimide) or N,N'-carbonyldiimdazole or an isoxazolium salt, for example, Woodwards reagent, the amino group for instance by reaction with a phosphite. As the most frequently used methods there may be mentioned the carbodiimide method, the Weygand-Wiiensch method (carbodiimide in the presence of N-hydroxysuccinimide), the azide method, the method of the activated esters and the anhydride method, also the Merrified method and the method of the N-carboxyanhydrides or N-thiocarboxyanhydrides.

Apart from the manufacture of the final products the invention includes also as a special object the manufacture of the starting materials, especially of the peptide fragment containing the disulphide bridge and its linking with the remainder of the peptide. When the aminoacid in position 10 to 12 is glycine, it is advantageous to start from a sequence that comprises the first 10 or 12 N-terminal aminoacids and to condense the whole remaining sequence with this N-terminus.

It is also possible to link the said N-terminal sequence with the fragment up to the 28th aminoacid (glycine) containing a free C-terminal carboxyl group and condense the octacosa-peptide with the tetrapeptide of the aminoacids 29-32. This condensation is performed, for example, by the Weygand-Wiinsch method. When the condensation of the sequence 1-10 or 1-12 is carried out with the C-terminal sequence 11-32 or 13-32, the carbodiimide method or the Weygand-Wiiensch is preferred.

, In the following preferred variants of the manufacture of the peptide of the formula I are explained in which the 8th aminoacid, methionine, is replaced by valine, norvaline, leucine, isoleucine, norleucine or a-aminobutyric acid (this 8th aminoacid is represented by X in the Figures). The diagrams illustrate special working methods which, of course, can be replaced by equivalent methods.

(3): the method of the activated esters, especially p-nitrophenyl ester (ONP) or hydroxysuccinimide ester (OSU) (4): the carbodiimide method (5): the Weygand-Wiiensch method BOC: tertiary butyloxycarbonyl DPC: 2- (p-biphenylyl) -isopropoxycarbonyl Z: carbobenzoxy TRI: trityl Bzl: benzyl OtBu: tertiary butyl ester OBzl: benzyl ester ONB: p-nitrobenzyl ester ONP: p-nitrophenyl ester OMe: methyl ester OEt: ethyl ester OCP: 2,4,5-trichlorophenyl ester tBu: tertiary butyl ether Ac: acetyl TFA: trifiuoracetic acid Abu: a-aminobutyric acid The N-terminal decapeptide (1-10), in which X has the meaning defined above, as the 8th aminoacid can be synthesized, for example, from the sequences 1-4 and 5-10, or 1-5 and 6-10, or 1-6 and 7-10, or 1-7 and 8-10, as shown in FIGS. 1 to 8, but it is also possible to use other fragments for synthesizing the sequence 1-10. The protective group for the a-amino group on cysteine 1 is preferably the tertiary butoxycarbonyl group or an equivalent group eliminable by acid hydrolysis, or, when an N-acylated dotriacontapeptide is to be manufactured, the corresponding acyl, for example acetyl, group. Apart from this it is advantageous to use mercapto protective groups that can be eliminated selectively with respect to the N- amino protective group (for example tertiary butyloxycarbonyl group) eliminable by acid hydrolysis, for instance the benzyl or trityl group. The terminal carboxyl group of the decapeptide need not necessarily by protected, for example it needs no protection when the condensation is performed by the azide or anhydride method. It is also possible to protect this group by esterification as indicated above, for example by esterification with methanol or ethanol (elimination of the ester group with dilute sodium hydroxide solution) or with benzyl alcohol or an analogue (elimination of the ester group, for example), with sodium in liquid ammonia). The amino groups of the intermediates are protected with the usual protective groups, for example carbobenzoxy, trityl, tertiary butyloxycarbonyl or 2-para-diphenyl-isopropyloxycarbonyl. The carboxyl groups of the intermediates are, if necessary, esterified in the usual manner. The hydroxyl groups of the serine and threonine residue can be protected by etherification, for example with tertiary butanol or an equivalent thereof.

The p-nitrobenzyl ester and benzyl ester groups can be eliminated with sodium in liquid ammonia or by hydrogenolysis in the presence of palladized carbon, the carbobenzoxy group likewise by hydrogenolysis; the N-trityl group with aqueous acetic acid, the tertiary butyloxycarbonyl group with trifiuoracetic acid, the 2 (p biphenylyl) isopropoxycarbonyl group with aqueous acetic acid or, for example, with a mixture of glacial acetic acid, formic acid of 82.8% strength and water (72122), as described in Kamber and Rittel, Helv. Chim. Acta 51 (1968), 2061. The p-nitrobenzyl or methyl ester can be converted into the hydrazide with hydrazine hydrate. The methyl or ethyl ester group is hydrolyzed with dilute sodium hydroxide solution. The tertiary butyl ester is split with trifiuoracetic acid, as is the tertiary butyl ether. The S-trityl groups are eliminated with mercuric acetate and sulphuretted hydrogen, the S-benzyl group with sodium in liquid ammonia, and any benzyl or p-nitrobenzyl ester groups are simultaneously eliminated. The cyclization to the disulphide is carried out, for example, by oxidation 8 with 1,2-diiodoethane, the cyclization of the S-tritylated compounds with iodine in methanol.

The C-terminal sequence comprising the aminoacids 11 to 32 or 11 to 28, to be linked with the N-terminal sequence is synthesized, for example, from the sequences 11-16, 17-20, 21-28 and 29-32, as shown in FIG. 9.

In the diagrams of FIG. 9 the hydroxyl groups of the threonine residues and of the tyrosine residue are protected, but this is not absolutely necessary. It is possible to link up also other sequences and to use other protective groups.

FIG. 10 illustrates the synthesis of the hexapeptide (in form of the hydrazide) of aminoacids 11-16. It can be linked with the sequence 17-28 or 17-32 by the azide method.

The sequence 17-28 can be synthesized from the fragments 17-20 and 21-28 by the azide method.

FIG. 11 illustrates the synthesis of the tetrapeptide hydrazide of the aminoacids 17-20. FIG. 12 shows the synthesis of the octapeptide 21-28. After having linked up the two sequences, the a-amino protective group is eliminated (the carbobenzoxy group by hydrogenolysis in the presence of palladium carbon) and the resulting dodecapeptide containing protected side-chains is condensed with the hexapeptide hydrazide 11-16 by the azide method (FIG. 10).

The resulting sequence 11-28 can be linked with the tetrapeptide amide of the aminoacids 29-32 which is manufactured as shown in FIG. 13, for example by the Weygand-Wuensch method; this gives rise to the protected docosapeptide amide 11-32, from which the a-amino protective group can be eliminated (carbobenzoxy for instance hydrogenolytically, DPC with acetic acid or glacial acetic acid 82.8% formic acid water (721:2), and the resulting compound containing a free a-amino group canafter having removed the acetic acid-be linked with the N-terminal decapeptide (FIGS. 1-8), for example by the method of the mixed anhydrides, of the activated esters (OSU) or according to Weygand-Wuensch.

Alternatively, after elimination of its a-amino protective group, the sequence 11-28 can be linked with the N-terminal decapeptide by the method of the mixed anhydrides, and the resulting product can be condensed with the tetrapeptide amide 29-32, for example according to Weygand-Wuensch.

Another way of synthesizing the C-terminal sequence 1l-32 is, for example, to synthesize it from the partial sequences 11-19 and 20-32, as shown in FIGS. 14 and 15, preferably by the method of the mixed anhydrides or according to Weygand-Wuensch.

From the protected dotriacontapeptide amide the protective groups are eliminated, for example with trifluoracetic acid, or with concentrated hydrochloric acid.

The dotriacontapeptide containing free or trityl-protected mercapto groups, to be used in variant (2) of the present process can be prepared as described above for the protected dotriacontapeptide, with the dilference that the protected mercapto groups are retained to the end of the synthesis. Only when all other protective groups have been eliminated from the protected dotriacontapeptide are the mercapto protective groups eliminated or the tritylated compound is oxidized directly as mentioned above.

The synthesis according to variant (3) of the present process is specially suitable for the manufacture of final products containing acylated amino groups and in the first place also for the manufacture of a-desaminopeptides or a-acylated peptides in which the aminoacid 18, lysine, is exchanged for an aminoacid whose side-chain contains no acylatable amino group, for example for glycine or for the aminoacid 18 of porcine thyrocalcitonine, asparagine. The Not-acylated decapeptide can be manufactured for instance as shown in FIG. 5 K, though it is also possible to use from the start the acyl group to be retained as amino protective group. The synthesis methods correspond to those described above.

Depending on "the 'reactionconditions employed the new compounds are obtained in the'form of bases or of phuricor phosphoric-acids, or with organic acids such as.

formic, acetic, propionic, glycollic, lactic, succinic, pyrruvic, oxalic, maloni'c, maleic, fumaric, malic, tartaric, citric, ascorbic, hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4 aminobenzoic, 4 hydroxybenzoic, anthranilic, cinnamic, mandelic, salicylic, 4 aminosalicylic, 2 -phenoxybenzoic, '2-- acetoxybenzoic, methanesulphonic, ethanesulphonic, hydroxyethanesulphonic, benzenesulphonic, p-toluenesulphonic, naphthalenesulphonic' or sulphanilic acid.

The peptides obtained by the present process can be used in form of pharmaceutical preparations which contain the peptides in conjunction or admixture with a pharmaceutical organic or inorganic excipien t suitable for enteral or parenteral administration. Suitable excipients are substances that do not react with the polypeptides, for example gelatin, lactose glucose, sodium chloride, starches, magnesium stearate, talcum, vegetable oils, benzyl alcohols, gums, polyalkyleneglycols, white petroleum jelly, cholesterol or other known medicinal excipients. The pharmaceutical preparations may be, for example, lyophilizates or in liquid form solutions, suspensions or emulsions. They may be sterilized and/or contain assistants such as preserving, stabilizing, wetting or emulsifying agents. They may also contain further therapeutically valuable substances.

The following Examples illustrate the invention.

In thin-layer chromatography the following systems are used:

43A: tertiary 'amyl alcohol+isopropanol+water 100:40: 10) 43C: tertiaryamyl alcohol-{-isopropanol-l-water (51:21:28) secondary butanol+3% aqueous ammonia (:30) 52: n-butanol+ glacial acetic acid+water 27.5 :21) 52A: n-butanol+glacial acetic acid+water (67:10:23) 79: n-butanol-l-pyridine-f-water (34:33:33)

- '87: isopropanol-l-glacial acetic acid+water (77:4:19)

89: ethyl acetate+acetone+water (72:24:4)

100: ethyl acetate+pyridine+ glacial acetic acid+water 102E: ethyl acetate+methylethylketone+glacial acetic acid+water (50:30:10z10) 107: ethyl acetate+pyridine+water (49:24:27)

115: ethyl acetate-l-pyridine-l-formic acid+water 121A: isopropanol+26% ammonia+water :5 :10)

system 1: benzene+ethanol (80:20)

system 2: benzene+ethanol (:10)

system 4: n-amyl alcohol-+formic acid+water system 5 n-butanol-glacial acetic acid+water system 6: n-butanol-pyridine-glacial acetic acid+water system 7: n-amyl alcohol+pyridine+water (70:20:10)

system 8: chloroformzmethanol:glacial acetic acid system 9: benzene+ethanol (70:30)

system 10: benzene+ethanol (60:40)

Thin-layer chromatography is performed on silica gel or alumina (A1ox D-O from Messrs. Camag with 8% gypsum) or on cellulose (Selecta 1440 from Messrs. Schleichter and Schuell).

'4 2 5 4 5 6 7 e 9 4o Cys Gly Asn Leu Ser Thr Cys X Iieu Gly r1 Z]. A 1300 on BOC-0H soc-on a n n z Boo-on uoma aoc H Boo-on BOC-OH HOBz1 1a eoc l 1t n -z Boc -oma Boc l--'oBz1 c n N2H2-Z H ONB n 01321 n Boc-- L" 112E245 Boc 01321 E 1300 N n H--- --OBzl B21 7 r BOC ons soc I OBzl Bzl c H ONB P OBzl a BOC one lizl Fig. 1 I 1300 2H5 i zl Bzl .r BOC I 1) l OBzI sit n K soc 21-:

L 1300 I DH 500 mg. of finely powdered Ala-Ile- Gly-Val- Gly-Ala-Pro-NH; are vigorously stirred into 10 m1. of ice-cold concentrated hydrochloric acid. After 10 minutes at C. 50 ml. of water and ml. of glacial acetic acid are added and the solution is freed from chlorine ions by filtration through a column of Merck ion exchange resin NlI, weakly basic, acetate form, the filtrate is mixed with ooctanol and evaporated to dryness; the residue is freed from octanol by being repeatedly washed with petroleum ether and decantation, dried, dissolved in 0.1 N-formic acid and purified by chromatography on a column (3.8 x 120 cm.) of Bio-Gel P Fractions of ml. each are collected and checked for purity by thin-layer chromatography on alumina (systems 52, 79 and 45), and the pure fractions are combined and lyophilized. In the thin-layer chromatogram on silica gel Rf =0.44; on alumina Rf =0.55; Rf =0.64; Rf =0.45.

The starting material can be prepared thus:

( 1 Z-Ala-Pro-OtBu 68.85 g. of Z-Ala-OH are dissolved in 550 ml. of dimethylformamide after which 43.3 ml. of triethylamine are added. At C. 30.8 ml. of ethylchloroformate are added and in 10 minutes the temperature is allowed to rise to 10 C.

Subsequently a solution of 52.65 g. of H-Pro-OtBu in 105 ml. of dimethylformamide is cooled to -10 C. and added.

After 30 minutes of stirring at 10 and 2 /2 hours at C. the solution is stored overnight in a refrigerator after which the solvent is evaporated in vacuo at a temperature of 4 0 C. The residue is taken up in aqueous ethylacetate, the solution washed with 0.1 N hydrochloric acid, 10% sodium chloride solution, 5% sodium bicarbonate and again sodium chloride solution and dried over sodium sulphate, then evaporated to dryness in vacuo. Rf 0.6.

(2) H-Al-Pro-OtBu, acetate 109 g. of Z-Ala-Pro-OtBu are dissolved in 649 ml. of methanol and the solution is hydrogenated in the presence of 4.1 ml. of glacial acetic acid and 3.1 g. of palladium carbon (10%). After filtration and evaporation of the solution to dryness the Rf value of the product is 0.5.

(3 Z-Gly-Ala-Pro-OtBu 56.75 g. of Z-Gly-OI-I are dissolved in 675 ml. of tetrao (4) H-Gly-Ala-Pro-OtBu 80 g. of Z-Gly-Ala-Pro-OtBu are dissolved in 800 m1. of methanol and hydrogenated as described under 2 above, but without acetic acid.

In the thin-layer chromatogram on silica gel the Rf value of the product is 0.30.

(5) Z-Val-Gly-Ala-Pro-OtBu 54.7 g. of I-I-Gly-Ala-Pro-OtBu are suspended in 722 ml. of ethylacetate. After cooling to -10 C. 71.6 g. of Z-Val-ONP are added. The mixture is stirred for 1 hour at -10 C., 1 hour at 10 C. and 20 hours at 20 C. The reaction mixture is washed with 0.1 N hydro- 22 chloric acid, salt water, 20% potassium carbonate and again with saline. The organic layer is dried and the solvent distilled oif in vacuo. The residual oil is washed with ethylacetate-hexane.

In the thin-layer chromatogram on silica gel Rf =0.4.

(6) Z-Val-Gly-Ala-Pro-OH 102 g. of Z-Val-Gly-Ala-Pro-OtBu are dissolved in 690 'ml. of dry ethylacetate after which a current of hydrochloric acid gas is bubbled through for 1% hours. The solvent is evaporated and the residue redissolved in ethyl acetate. .After the addition of hexane and stirring for 1 hour at -10 C. the suspension is filtered. The product melts at 111 C. (with decomposition) [a] =-38.3 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.50.

7) Z-Val-Gly-Ala-Pro-NH 64.4 g. of Z-Val-Gly-Ala-Pro-OH are dissolved in 644 ml. of dry tetrahydrofuran. The mixed anhydride is formed with 18.9 ml. of triethylamine and 13.5 ml. of ethylchloroformate as described under 1 above. 25 ml. of liquid ammonia are added and the reaction mixture is worked up as described under 1. The product melts at 208-211 C. [a] =-32.7 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.7.

(8) H-Val-Gly-Ala-Pro-NH sulfate 27.5 g. of Z-Val-Gly-Ala-PrdNH are suspended in 550 ml. of methanol and after adding of 3 ml. of 96% sulfuric acid hydrogenated as described under 2 above. In the thin-layer chromatogram on silica gel the product has an Rf value of 0.2.

(9) Z-Ile-Gly-OMe 84.2 g. of H-Gly-OMe and 247 g. of Z-Ile-ONP are suspended in 2.4 liters of dimethylformamide, and after cooling to --10 C. 99 ml. of triethylamine are added. The mixture is worked up as described under 5 above. The product crystallizes from ethyl acetate-hexan. Melting point 126128 C. [a] 27 (c.=3 in methanol). In the thin-layer chromatogram on silica gel Rf =0.50.

(l0) H-Ile-Gly-OMe, sulfate 193 g. of Z-Ile-Gly-OMe are dissolved in 4860 ml. of methanol and 30 ml. of 96% sulfuric acid. Hydrogenation is carried out as described under 2 above. In the thin-layer chromatogram on silica gel Rf =0.56.

11 Z-Ala-Ile-Gly-OMe 125.3 g. of Z-Ala-OH are dissolved in 1250 ml. of dimethylformamide. The mixed anhydride is prepared with 79.3 ml. of triethylamine and 55.7 ml. of ethylchloroformate and allowed to react with a solution of 168 g. of H-Ile-Gly-OMe, sulfate, in 835 ml. of dimethylformamide and 158.3 ml. of triethylamine as described under 1 above. The dimethylformamide is eliminated under reduced pressure to ml. and the solution poured into 500 ml. of water. The product melts at 191.5-l92.5 C. [a] 9.2 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rj =0.65.

(12) H-Ala-Ile-Gly-OMe, /2 1-1 30 100 g. of Z-Ala-Ile-Gly-OMe are suspended in 824 ml. of dimethylformamide and 6.66 ml. of 96% sulfuric acid and hydrogenated as described under 2 above. In the thin-layer chromatogram on silica gel Rf =0.5.

(13 BOC-Thr-Ala-Ile-Gly-OMe 57.8 g. of BOC-Thr-NH-NH are dissolved in 340 ml. of dimethylformamide and the solution cooled to 20 C. 250 ml. of 1.989 N-hydrochloric acid and then 33.5 ml. of iso-amyl-nitrite are added to the solution which is then stirred vigorously for 10 minutes. In the meantime,

105 ml. of triethylamine are added to a solution, cooled to 10 C., of 79.1 g. of H-Ala-Ile-Gly-OMe in 1500 ml. of dimethylformamide. This solution is cooled to -20 C. and the above solution of BOC-Thr-N is added. The mixture is allowed to react for 70 hours at C., the solvent is evaporated ofi? at 40 C. under reduced pressure, the residue dissolved in ethyl acetate, the solution washed with 0.1 N hydrochloric acid, water, sodium bicarbonate and water, then dried. The ethyl acetate is then removed under reduced pressure and the residue crystallised from ethyl acctate/ hexane. The product melts at l87l89 C. [u] 13.2 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf ==0.68.

(l4) BOC-Thr-Ala-Ile-Gly-NH-NH 87 g. of the BOC-tetrapeptidester are dissolved in 870 ml. of methanol after which the solution is cooled to 0 C. and 37.6 ml. of hydrazin hydrate are added. After 18 hours at room temperature the suspension was stirred for 1 hour at 0 C. and filtered. The residue is crystallised twice from methanol. Melting point 231-2325 C. [a] 5.9 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rfq=0.7.

( l 5 BOC-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH 27.1 g. of BOC-Thr-Ala-Ile-Gly-NH-NH are suspended in 250 ml. of dimethylformamide. The azide is prepared with 59.9 ml. of 2.23 N hydrochloric acid and 9.1 ml. of isoamylnitrite. The solution is stirred for minutes.

24.2 g. of l-l-Val-Gly-Ala-Pro-NH sulfate, are dissolved in 120 ml. of dimethylformamide and after cooling to 0 28 ml. of triethylamine are added. After mixing with the azide the pH is brought up to 7. The mixture is allowed to react for 70 hours. The peptide is crystallised from ethylacetate/hexane. Melting point 226-2275 C. [a] =25.6 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.7.

( l 6) H-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH trifiuoracetate 51 g. of BOC-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH are dissolved in 153 ml. of 90% aqueous trifiuoro acetic acid. The solution was left for 1 hour and then 1530 ml. of dry ether are added. The precipitate obtained is filtered and washed three times with ether. Melting point 221- 223 C. In the thin-layer chromatogram on silica gel Rf =0.5.

( 17) Z-Pro-Gln-NH-NH-BOC 67.23 g. of Z-Pro-OH are dissolved in 670 ml. of dimethylformamide. Reaction with 38.9 ml. of triethylamine and 26 ml. of ethylchloroformate furnishes the mixed anhydride.

70.2 g. of H-Gln-NH-NH-BOC are allowed to react with this anhydride as described under 1 above. The residue is crystallised from hot ethylacetate. Melting point 169-171 C. [0:1 :3 41.3 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.45.

( 1 8) H-Pro-Gln-NH-NH-BOC 94 g. of the derivative described under 17 above are dissolved in 564 ml. of dimethylformamide and the solution is hydrogenated as described under 2, but without acid. In the thin-layer chromatogram on silica gel (19) Z-Phe-Pro-Gln-NH-NH-BOC 80.4 g. of Z-Phe-ONP, 68 g. of H-Pro-Gln-NH-NH- BOC and 9.4 ml. of glacial acetic acid are dissolved in 560 ml. of dimethyl formamide as described under 5 above. The product melts at 107 C. [a] 35.9 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.7.

24 2o H-Phe-Pro-Gln-NH-NH-BOC 103 g. of Z-Phe-Pro-Gln-NH-NH-BOC are dissolved in 618 ml. of dimethylformamide and hydrogenated as described under 2 above, but without acid. In the thin-layer chromatogram on silica gel the product has an Rfg value of 0.5.

21 Z-Thr-Phe-Pro-Gln-NH-NH-BOC 46.1 g. of Z-Thr-NI-I-NH are suspended in 550 m1. of dimethylformamide. The azide is prepared with 177 ml. of 1.958 N hydrochloric acid in tetrahydrofuran and 24 ml. isoamylnitrite. The azide is coupled with 81.4 g. of H- Phe-Pro-Gln-NH-NH-BOC dissolved in 1200 ml. of dimethylformamide and 48.5 ml. of triethylamine. After crystallization from ethyl acetate+hexane the product melts at l29-134 C. [a] =34.8 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.85.

(22) H-Thr-Phe-Pro-Gln-NH-NiH-BOC 23.6 ml. of the tetrapeptide described under 21 are dissolved in 188 ml. of dimethylformamide and hydrogenated in the absence of acid. In the thin-layer chromatogram on silica gel Rf =O.7.

(23) Z-His-Thr-Phe-Gln-NH-NH-BOC 9.68 g. of Z-His-NH-NH are dissolved in 100 m1. of dimethylformamide. The azide prepared with 36.3 ml. of 2.67 N hydrochloric acid in tetrahydrofuran and 4.32 ml. of isoamylnitrite is coupled with 19.32 g. of H-Thr-Phe- Pro-Gln-NH-NH-BOC dissolved in 300 ml. of dimethylformamide and 13.8 ml. of triethylamine. The reaction mixture is filtered, evaporated in vacuo to 100 ml. and poured into 1100 ml. of ethylacetate. The product melts at 95133 C. In the thin-layer chromatogram on silica gel Rf =0.7.

(24) Z-His-Thr-Phe-Pro-Gln-NH-NH HCl 11.4 g. of Z-His-Thr-Phe-Pro-Gln-NH-NH-BOC are dissolved in 57 ml. of trifiuoro acetic acid. The solution is left to stand for 1 hour and then 9.8 ml. of 4 N hydrochloric acid are added. The solution is poured into 570 ml. of dry ether. The residue is dried in vacuo over potassium hydroxide. In the thin-layer chromatogram on silica gel Rf =0.25.

(25) Z-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly- Val-Gly-Ala-Pro-NH 11 g. of the hydrazide described under 24 above are dissolved in 47 ml. of dimethylformamide. The azide is prepared with 15.6 ml. of 2.267 N hydrochloric acid and 1.57 ml. of amylnitrite as described under 13 above. The azide is allowed to react with 6 g. of the octapeptide amide described under 16 above, dissolved in 70 ml. of dimethylformamide and 5.5 ml. of triethylamine. The pH is 6.5. After 7 days the reaction mixture is filtered, the filtrate poured into 710 ml. of ethyl acetate and the residue stirred twice with ethyl acetate. The product melts at 186 C. with decomposition. In the thin layer chromatogram on silica gel Rf -;=0.6.

(26) Z-Lys (BOC) -Phe-OBzl 261.6 g. of Z-Lys(BOC)OH dicyclohexylammonium salt are suspended in 6.4 ml. of acetonitrile, 137.6 g. of H-Phe-OBzLHCl are added and this suspension is stirred for 30 minutes. The mixture obtained is cooled to 5 after which 96 g. of dicyclohexylcarbodiimide are added. The suspension is stirred for 20 hours at room temperature, filtered and the residue washed with acetonitrile. The filtrate is evaporated in vacuo and the residue dissolved in ethylacetate, the solution washed with 0.1 N hydrochloric acid, water, 5% sodium bicarbonate and water. After drying the solvent is evaporated and the residue crystallised from ethylacetate/hexane. Melting point 101- 103 C. [a] =12..5 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.55.

25 (27)H-Lys(BOC-Phe-O'H, HCl

105.4 g. of the above dipeptide ester are dissolved in 630 ml. of dimethylformamide and the solution hydrogenated as described under 2 above, but without glacial acetic acid. However, 150ml. of 1.15 N hydrochloric acid in tetrahydrofuran are added after 2% hours. The hydrogenation takes 20 hours. In the thin-layer chromato gram on silica gel the product has an Rf value of 0.7.

(28) Z-Asn-Lys(BOC)-Phe-OH 76.2 g. of Z-Asn-ONP and 73.6 g. of H-Lys(BOC)-Phe- OH.HCl are dissolved in 850 ml. of dimethylformamide and 150 ml. of tetrahydrofuran. After cooling to 10 C. 47.8 ml. of triethylamine are added as described under 5. After a reaction time of 20 hours at 20 C. the reaction mixture is evaporated to 320 ml. of solvent and poured out into 1435 ml. of ethyl acetate and 359 ml. of 0.1 N hydrochloric acid. The precipitate is washed with water and ether. Melting point 167-168 C.

(c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf7=0-7.

(29) H-Asn-Lys(BOC)-Phe-OH, HCl

34 g. of the above product are dissolved in 240 ml. of methanol and the solution hydrogenated as described under 27, except that 13.3 ml. of 2 N-hydrochloric acid in tetrahydrofuran are added after 2% hours and another 13.3 ml. of hydrochloric acid in tetrahydrofuran after 20 hours. The solution is processed as it is. In the thin-layer chromatogram on silica gel Rf =0.6.

(30) Z-Asp (OtBu) -Phe-O Me 100.84 g. of Z-Asp(OtBu)-OH, dicyclohexylammonium salt are dissolved in 880 ml. of methylene chloride, 43.16 g. of H-Phe-OMeHCl are added and stirring is continued for 10 minutes. The suspension is cooled to 5 C. after which 41.2 g. of dicyclohexylcarbodiimide in 120 m1. of methylene chloride are added and the batch is worked upon up as described under 26 above. The residue is washed with methylene chloride. In the thin-layer chromatogram on silica gel Rf =0.9'.

(3 1 Z-Asp OtBu) -Phe-NH-NH 79.2 g. of the above dipeptide ester are dissolved in 1320 ml. of methanol, the solution is cooled to C., and 21.1 ml. of hydrazine hydrate added as described under 14 above, but the reaction is allowed to proceed for 3 days. The residue is washed twice with water. Melting point 143-145 C. [a] =-22.2 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.65.

(3 2) Z-Asp OtBu) -Phe-Asn-Lys (BOC) The-OH 54.5 g. of Z-Asp(OtBu)-Phe-NH-NH are dissolved in 325 ml. of dimethylformamide. The azide is prepared as described under 13 above with 111 m1. of 1.98 N hydrochloric acid in tetrahydrofuran and 14 ml. of isoamylnitrite. 53.4 g. of H-Asn-Lys(BOC)-Phe-OH.HC1 are dissolved in 280 ml. of dimethylformamide and 50 m1. of triethylamine and added to the azide solution after cooling to -10 C.

The pentapeptide is precipitated by mixing the residue with ethyl acetate and washed with water-l-ethanol l: 1. Melting point 189-190 C. [a] =21.8 (0:1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.77.

(3 3) H-Asp(OtBu) -Phe-Asn-Lys(BOC) Phe-OH 9.6 g. of the protected pentapeptide obtained as described under 32 above are dissolved in 192 ml. of dimethylformamide and the solution hydrogenated as described under 27 above, except that 4.43 ml. of 2.3

26 N-hydrochloric acid in tetrahydrofuran are used. In the thin-layer chromatogram on silica gel the product has an Rf value of 0.8.

(34) BOC-TyrThr-ONB 69.68 g. of *H-Thr-ONHHCI are suspended in 480 ml. of acetonitrileand 32.84 ml. of triethylamine are added. The mixture obtained is cooled to 5 C. followed by adding of 67.52 g. of BOC-Tyr-OH and 960 ml. of cooled acetonitrile. After adding 49.44 g. of dicyclohexylcarbodiimide the suspension is stirred for 1 hour at 3 C. and worked up as described under 26 above. The dipeptide is isolated as an oil. In the thin-layer chromatogram on silica gel Rf =0.60.

(35) H Tyr-Thr-ONB-HCI 121 g. of BOC-Tyr-Thr-ONB are dissolved in 1537 ml. of methylene chloride and 384 ml. of nitromethane and the further procedure described under 6 above is followed. HCl gas is passed through for 45 minutes and the suspension then stirred for 1 hour before it is filtered. The product melts at 226-227 C. In the thin-layer chromatogramon silica gel Rf =0.35.

(36) Z-Thr-Tyr-Thr-ON-B 57.4 g. of Z-Thr-NH-NH are suspended in 320 ml. of dimethylformamide and the azide formed with 220 ml. of 1.953 N-hydrochloric acid in tetrahydrofuran and 29 ml. of isoamylnitrite as described under 13 above. The azide solution is added to a solution of 98 g. of H-Tyr-Thr-ONB, HCl in 760 ml. of dimethylformamide and 91 ml. of triethylamine. The tripeptide derivative crystallizes from ethyl acetate-l-hexane. Melting point 111-118 C. [a] =4-.4 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf1=0.6.

(37) Z-Thr-Tyr-Thr-NH-NH 114.2 g. of the above tripeptide ester are dissolved in 480 ml. of methanol. 48 ml. of hydrazine hydrate are added.

After 4 hours 1440 ml. of ethyl acetate are added and after 1% hours the suspension is filtered. The residue is stirred for 20 hours with 1 l. of ethyl acetate. The product melts at 2l9221 C. [u] =6.6 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.85.

89 g. of Z-Thr-Tyr-Thr-NH-NH are suspended in 890 ml. of dimethylformamide. The azide is formed as described under 13 above with 197 ml. of 2.02 N hydrochloric acid in tetrahydrofuran and 26.8 ml. of isoamylnitrite and added to 39.3 g. of H-Gln-NH-NH-BOC and 56.2 ml. of triethylamine in 750 ml. of dimethylformamide cooled to -10 C. Reaction time: 4 days. After evaporating of the reaction mixture 720 ml. of methanol and 720 ml. of ether are added and the suspension stirred for 2 hours. The crystals are then filtered off and stirred with 400 ml. of 0.05 N-hydrochloric acid (twice). The product melts at 216-218" C. [u] =-8.3 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.8.

53.5 g. of the derivative described under 38 above are dissolved in 110 ml. of trifluoracetic acid, the solution is stirred for 1% hours, then treated with 880 m1. of ether as described under 16 above. The precipitate is stirred with 200 ml. of methanol-l-ether (1:4) (twice). The products melts at 200-203 C. [a] -4.6 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.75.

27 40) Z-Thr-Tyr-Thr-Gln-Asp OtBu) Phe-Asn- Lys (BOC Phe-OH 9.5 g. of Z-Thr-Tyr-Thr-Gln-NH-NH are suspended in 95 ml. of dimethylformamide, and the azide is formed with 11.94 ml. of 2.122 N-hydrochloric acid in tetrahydrofuran and 2.28 ml. of isoamylnitrite as described under 13 above. 8.6 g. of H-Asp(OtBu)-Phe-Asn-Lys- (BOC)-Phe-OH are dissolved in 210 ml. of dimethylformamide and 7.5 ml. of triethylamine. This solution is cooled to 10 C. and added to the azide solution. The reaction is allowed to proceed for 3 days at C. The batch is then filtered, the filtrate evaporated, and the residue stirred with 300 ml. of 0.1 N-hydrochloric acid. The product crystallizes from dimethylformamide and 0.1N-hydrochloric acid 1:3.5). It melts at 206 C. [a] =19.3 (0.:1 in dimethylformamide). In the thin layer chromatogram on silica gel Rf =0.87.

(41 H-His-Thr-Phe-ProGln-Thr-Ala-Ile-Gly- Val-Gly-Ala-Pro-NH 32 g. of the BOC-tridecapeptideamide described under 25 above are dissolved in 1200 ml. of dimethylformamide and 12 ml. of 4 N-hydrochloric acid, and the solution is hydrogenated for 20 hours as described under 2 above. The filtrate is concentrated to 250 ml. and stirred with 1500 ml. of dry ether. The crude product is purified by counter-current distribution according to Craig in the system n-butanol-l-glacial acetic acid+water (411:5). K=0.11. [a] =39.4 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.2.

(42) Z-Thr-Tyr-Thr-Gln-Asp(OtBu) Phe Asn Lys (BOC)Phe-His-Thr-Phe-Pro-Gln-Thr-Ala Ile Gly- Val-Gly-Ala-Pro-NH 7.3 g. of the protected nonapeptide described under 40 above are dissolved in 75 ml. of dimethylformarnide. 1.33 g. of N-hydroxysuccinimide are added, and the solution cooled to 22 C. A solution of 1.195 g. of dicyclohexylcarbodiimide in ml. of dimethylformamide, likewise cooled to 22 C., is added (solution A). After stirring at 22 C. for 1 hour and at +4 C. for 5 hours, the suspension is again cooled to 22 C. A solution of 6.9 g. of the tridecapeptideamide described under 41 in 80 ml. of dimethylformamide is given a pH of 6.7 to 7 and cooled, then added to solution A. After a reaction period of 14 days the suspension is added to a fresh solution A, prepared from 0.73 g. of the nonapeptide described under 40 above. The reaction is allowed to proceed for another 7 days at 0 C., the suspension then filtered, and the filtrate evaporated to dryness under reduced pressure. The residue is purified by chromatography through a column of silica gel using the system n-butanol-i-glacial acetic acid+water (4:1:5). In the thin-layer chromatogram on silica gel the product has an Rf value of 0.32.

(43) H-Thr-Tyr-Thr-Gln-Asp(OtBu) Phe Asn-Lys- (BOC)Phe-His-Thr-Phe-Pro-Gln-Thr-Ala Ile Gly- Val-Gly-Ala-Pro-NH 2 g. of the product described under 42 above are dissolved in 150 ml. of dimethylformamide and hydrogenated. After 2% hours 0.3 ml. of 4.844 N hydrochloric acid are added. After 21 hours the solution is filtered and the filtrate is evaporated.

The residue is dissolved in 25 ml. of dimethylformamide, 0.73 ml. of 1.0 N hydrochloric acid is added. The solution is added to 40 ml. of ethyl acetate and cooled to 0 C. After filtration the residue is taken up in tertiary butanol-water and lyophilised. [a] =25.2 (c.=2 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.32.

28 (44 BOC-Ser-Thr-OBzl 47.0 g. of H-Thr-OBZLHCI in 300 ml. of methylene chloride are added to a solution of 73.8 g. of BOC-Ser- OH. Dicyclohexylammonium salt in 500 ml. of methylene chloride and the mixture stirred for 10 minutes at room temperature and then cooled to 5 C. A solution of 40.1 g. of dicyclohexylcarbodiimide in ml. of methylene chloride is added dropwise at this temperature. The mixture is stirred for 3 hours at 5 C. and overnight at room temperature. After filtering off the dicyclohexylurea and dicyclohexylamine hydrochloride the solution is extracted by shaking three times with 0.1 N hydrochloric acid, twice with 20% strength sodium chloride solution, once with 10% strength sodium bicarbonate solution and twice with 20% strength sodium chloride solution and is dried over sodium sulphate. The solution is concentrated to about 600 ml., cooled to 5 C. and further dicyclohexylurea is filtered off. After evaporation to dryness, the residue is crystallised from ethyl acetate-hexane in order to purify it.

Melting point -111 C.; [a] =8.5 (c.=2 in dimethylformamide); Rf =0.33 (on silica gel).

(45) H-Ser-Thr-OBZLTFA 65.7 g. of BOC-Ser-Thr-OBzl are dissolved in 100 ml. of 90% strength trifiuoracetic acid and the solution is left for one hour at 20 C. It is then added dropwise to 1000 ml. of dry ether whilst stirring and the mixture is stirred for one hour and left to stand overnight at -10 C. The resulting precipitate is filtered off and washed three times with dry ether and dried in vacuo over sodium hydroxide; melting point 128-129 C.; Rf =0.65; Rf =0.50 (on silica gel).

(46) BOC-Leu-Ser-Thr-OBzl 52.5 g. of H-Ser-Thr-OBzLTFA are dissolved in ml. of dimethylformamide and mixed at 0 C. with a solution of 48.0 g. of BOC-Leu-ONP. 21 ml. of triethylamine and 0.75 ml. of glacial acetic acid are then added. The reaction mixture is stirred for 3 hours at 0 C. and overnight at room temperature. After adding 2.1 litres of ethyl acetate the solution is extracted by shaking twice with water, twice with 0.1 N hydrochloric acid, twice with 10% strength sodium chloride solution, eight times with 20% strength potassium carbonate solution, twice with 10% strength sodium chloride solution and once with 30% strength sodium chloride solution. After drying over sodium sulphate the solution is concentrated to about 1.4 litres. The protected tripeptide of melting point 114-116 C. crystallises overnight in the refrigerator; [a] =l4 (c.=2 in dimethylformamide); Rf =0.25 (on silica gel).

(47) H-Leu-Ser-Thr-OBzLTF A 46.1 g. of BOC-Leu-Ser-Thr-OBzl are dissolved in 92.5 ml. of 90% strength trifiuoracetic acid and left for 1 hour at 20 C. Thereafter dry ether (925 ml.) is added whilst stirring, and the mixture is stirred for one hour at 0 C. and left to stand overnight at 10 C. The resulting precipitate is filtered off, washed three times with dry ether and dried in vacuo over sodium hydroxide. Melting point 168171 C.; Rf =0.80 (on silica gel).

(48) BOC-Asn-Leu-Ser-Thr-OBzl 20.8 g. of BOC-Asn-OH are suspended in 208 ml. of acetonitrile, mixed with 22.7 g. of Woodwards Reagent K and the mixture stirred for 30 minutes at 20 C. 12.6 ml. of triethylamine are then added dropwise, whilst stirring, in such a way that the internal temperature does not exceed +32 C., and the mixture is cooled to 20 C. and stirred for a further 50 minutes at this temperature. The almost clear solution is cooled to 0 C. and is mixed with a solution, also cooled to 0 C., of 39.1 g. of H-Leu-Ser- Thr-OBZLTFA and 10.5 ml. of triethylamine in 257 ml.

of dimethylformamide. The reaction mixture, which soon solidifies, is stirred overnight at room temperature, cooled to 10 C. and stirred for a further 2 hours at 10 C. Thereafter the crystalline precipitate is filtered off and the tetrapeptide derivative is washed once with cold acetonitrile, once with ethyl acetate and three times with water until it is free of chloride. The resulting product is crystallised from 370 ml. of dimethylforrnamide, 3.7 ml. of glacial acetic acid and 370 m1. of acetonitrile. Melting point 225-226 C.; [(11 36 (c.=2 in dimethylformamide); Rf =0.85 (on silica gel).

32 g. of BOC-Asn-Leu-SenThr-OBZl are dissolved in 192 ml. of 90% strength trifiuoracetic acid and the solution left for 45 minutes at 20 C. Thereafter the solution is concentrated to about 40 ml., mixed with 400 ml. of ether whilst stirring and stirred for 20 minutes at 35 C. under reflux. The crystal suspension is then cooled to 10 C. and is left to stand overnight at -10 C. The precipitate is filtered off, washed three times with ether and dried in vacuo over sodium hydroxide. Melting point 125-127 C.; Rf =0.33 (on silica gel).

(50) BOC-Gly-Asn-Leu-Ser-Thr-OBzl 26.3 g. of H-Asn-Leu-Ser-Thr-OBZI.TFA are dissolved in 100 ml. of dimethylforrnamide, the solution is cooled to C. and successively mixed with 7.5 ml. of triethylamine, 0.54 ml. of glacial acetic acid and a solution of 14.4 g. of BOC-Gly-ONP in 100 ml. of dimethylformamide, stirred at room temperature until the mixture solidifies and left to stand for 2 days. Thereafter 300 ml. of ethyl acetate are added whilst stirring and the mixture left to stand overnight at 10 C. The precipitate is filtered 01f, washed twice with ethyl acetate and twice with ether, stirred for half an hour with 200 ml. of water, filtered off, washed with water and dried in vacuo. Melting point 227- 228 C.; [0611 -23 (c.=2 in dimethylformamide); Rf ==0.50 (on silica gel).

(5 1) BOC-Gly-Asn-Leu-Ser-Thr-OH 17.0 g. of BOC-Gly-Asn-Leu-Ser-Thr-OBzl are dissolved in 340 ml. of dimethylformamide whilst warming. After cooling to room temperature, 3.4 g. of strength palladium on charcoal are added and the product is hydrogenated. The reduction is complete after 4 hours. After filtering off the catalyst the solution is concentrated in a high vacuum. Triturating three times with ether yields a pentapeptide which is homogeneous according to a thin layer chromatogram. Melting point 181-183" C.; [a] 16.5 (c.=2 in dimethylformamide); Rf =0.65 (on silica gel).

9.2 g. of H-Cys(BZl)-N H -Z.HCl are dissolved in 300 ml. of freshly distilled dimethylforrnamide, mixed with 12.2 g. of BOC-Gly-Asn-Leu-Ser-Thr-OH and stirred at room temperature until solution has occurred. The solution is then cooled to 0 C., 3.28 ml. of triethylamine and 4.88 g. of N-hydroxysuccinimide in 100 ml. of dimethylformamide are added, the mixture is further cooled to 22 C. and 4.3 6 g. of dicyclohexylcarbodiimide in 30 ml. of dimethylformamide are added. The mixture is stirred for one hour at 22 C., the internal temperature is then allowed to rise gradually, the mixture stirred for a further 3 days at room temperature, the precipitated dicyclohexylurea filtered off and the filtrate evaporated to dryness in a high vacuum. The residue is stirred with a mixture of ethyl acetate and 5% strength citric acid solution and the precipitate is filtered off, washed with water, dried in vacuo, stirred with dry ether, filtered and dried in a high vacuum. Melting point 173-178 C.; [m] -25.5 (c.=2 in dimethylformamide); Rf =0.21 (on silica gel).

13 g. of BOC-Gly-Asn-Leu-Ser-Thr-Cys(Bzl)-N H -Z are dissolved in 130 ml. of Strength trifluoracetic acid and the solution is left to stand for 2 hours at 22 C. It is then concentrated and the residue stirred three times with ether and dried in vacuo over sodium hydroxide. Melting point 159-161 C. (Rf =0.63 (on silica gel).

(54) BOC-Cys(Bzl -Gly-Asn-Leu-Ser-Thr- Cys 'NzHg'Z 6.69 g. of BOC-Cys(Bz1)-OSU and 12.2 g. of H-Gly- Asn-Leu-Ser-Thr-Cys(Bzl)-N H -Z. 1.22 TFA are dissolved in ml. of dimethylformamide. Triethylamine is added dropwise from a solution of mMols of triethylarnine in 100 ml. of dimethylformamide, whilst stirring, until the reaction mixture shows a pH value of 6.4 on moist indicator paper. The solution is stirred for 3 days at room temperature and thereafter evaporated to dryness in a high vacuum. The residue is twice stirred with 300 ml. of ethyl acetate thereafter stirred three times with a mixture of 150 ml. of ethyl acetate and 30 ml. of 5% strength citric acid solution, filtered olf, dried in vacuo and crystallised from dimethylformamide-ethyl acetate. Melting point 191-193 C., [u] =31.5 (c. 2 in dimethylformamide); Rf =0.35 (on silica gel).

(55 BOC-Cys-Gly-Asn-Leu-Ser-Thr-Cys-N H 6 g. of BOC-Cys(Bz1)-Gly-Asn-Leu-Ser-Thr-Cys(B21)- NzHyZ are dissolved at 40 C. in 700 ml. of dry liquid ammonia. 973 mg. of sodium are added at the boiling point of the ammonia, whilst stirring, in such a way that the colour of the reaction mixture only becomes light blue. After 25 minutes the reduction is complete. The mixture is stirred for a further 10 minutes with the blue colouration being retained, 2.4 ml. of glacial acetic acid are then added and the mixture evaporated to dryness in a high vacuum (about 1 mm.). The residue is stirred with 12 ml. of water, 3.2 ml. of glacial acetic acid and 20 ml. of ethyl acetate for one hour at 0 C. and the precipitate is filtered off, twice washed with 10 ml. of 1% strength acetic acid solution and once with 10 ml. of ethyl acetate and dried in a high vacuum. Rf -=0.65 (on silica gel). (56) BOC-Cys-G1y-Asn-Leu-Ser-Thr-Oys-NzHa 1.0 g. of BOC-Cys-Gly-Asn-Leu-Ser-Thr-Cys-N H is dissolved in 100 ml. of dimethylformamide and 500 ml. of water containing 1.23 milliequivalent of hydrogen chloride. The pH-value of the solution is adjusted to 6.8 by means of 3.7 ml. of an 0.43 N potassium hydroxide solution. 247 ml. of an 0.01 M potassium ferricyanide solution and 4.9 ml. of an 0.43 N potassium hydroxide solution are simultaneously added dropwise with stirring over the course of 90 minutes whilst maintaining this pH-value. The mixture is stirred for a further 90 minutes at room temperature and the pH-value of the solution is then brought to 4.0 by means of 0.7 ml. of glacial acetic acid. The solution is thereafter stirred with 50 ml. of Dowex-2-X8, acetate form and subsequently with 13 ml. of Dowex-SOW-X8, H+ form, the mixture filtered and the filtrate evaporated to dryness. The residue is dissolved in 50% strength t-butanol, lyophilised and dried in a high vacuum. The product contains about 15% of potassium acetate. It is homogeneous in a thin layer chromatogram 31 (58) BOC-Nle-Leu-Gly-OEt As described under 1 above, the mixed anhydride is prepared from 4.63 g. of BOC-NleOH, 2.82 ml. of triethylamine and 1.9 ml. of ethylchloroformate in 50 ml. of tetrahydrofuran and coupled with 5.05 g. of H-Leu-Gly- OEt.HCl in 50 ml. of tetrahydrofuran.

The residue is crystallised from ethyl acetate-petroleum ether (40:60). Melting point 126-127 C. [a] 28.3 (c.=1 in dimethylformamide.) In the thin-layer chromatogram on silica gel Rf =0.7.

(59) BOC-Nle-Leu-Gly-OH 3.25 g. of the ester described under 58 above are dissolved in 100 ml. of methanol, and 21 ml. of 0.53 N sodium hydroxide are added. After stirring for 1 /2 hours the solution is acidified and evaporated in vacuo. The residue is dissolved in water and extracted with ethyl acetate. The organic layer is dried and evaporated. The product melts at 7172 C. [a] =28.4 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.75.

(60) H-Nle-Leu-Gly-OI-I 2.42 g. of BOC-Nle-Leu-Gly-OH are dissolved in 24 ml. of 90% trifluoracetic acid as described under 16 above. After 1 hour the solution is evaporated. The product melts at 9496 C. In the thin-layer chromatogram on silica gel Rf :0.56.

B O C-Cys- Gly-Asn-Leu-S er-Thr-C ys-Nle-Leu- Gly-OH 1.0 g. of the hydrazide described under 56 above is dissolved in 35 m1. of dimethylformamide. The azide is prepared as described under 13 above with 0.85 ml. of 3.64 N hydrochloric acid in tetrahydrofuran and 0.155 ml. of isoamylnitrite and added to a solution of 0.955 g. of H- Nle-Leu-Gly-OH and 0.324 ml. of triethylamine in 25 ml. of dimethylformamide. The reaction mixture is evaporated and twice stirred with ethyl acetate/citric acid solution. In the thin-layer chromatogram on silica gel Rf =0.7l.

BOC-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Nle-Leu-Gly-Thr-Tyr-Thr-Gln- Asp( OtBu) -Phe-As11-Lys(B O O)Phe-His-Thr-Phe-Pro-Gln-Thr- Ala-Ile-Gly-Val-Gly-Ala-Pro-NH:

440 mg. of the BOC-decapeptide described under 61 above and 109 mg. of N-hydroxysuccinimide are dissolved in 35 ml. of dimethylformamide. The solution is cooled to 22 C., '98 mg. of dicyclohexylcarbodiimide are added, and the whole stirred at 22 C. for 1 /2 hours and at C. for 3 /2 hours. The batch is then cooled to 22 C. and 850 mg. of the docosapeptideamide described under 43 are added, and the pH adjusted to 6.6 with triethylamine. After 7 days at 0 C. and days at 22 C. the solution is evaporated to dryness under reduced pressure and the residue chromatographed over silica gel using the system n-butanol-glacial acetic acid-water (4:1:5). In the thin-layer chromatogram on silica gel Rf =O.29.

EXAMPLE 2 70 mg. of

BOC-Cys-Gly-Asn-Leu-Ser-Thr-Gys-Val-Leu-Gly-Thr-Tyr-Thr-Gln- Asp(OtBu)-Phe-Asn-Lys(BOC)-Phe-His-Thr-Phe-Pro-Gln-Thr- Ala-Ile-Gly-Val-Gly-Ala-Pro-NH:

are dissolved in 3 ml. of ice-cold trifiuoracetic acid of 95% strength and the solution is heated to 25 C. After 1 hour at 25 C. the solution is poured into 70 ml. of icecold ether and the resulting precipitate is suctioned off, repeatedly washed with ether, dried and dissolved in 5% acetic acid. To remove trifluoracetate ions the whole is filtered through Merck ion exchange resin NII (weakly basic, acetate form). The eluate is lyophilized. The resulting dotriacontapeptide is not quite unitary according to its thin-layer chromatogram (alumina plates, systems 52 and 45); it can be purified by counter-current distribution in the system n-butanol-i-glacial acetic acid-l-water (411:5). In the thin-layer chromatogram on silica gel Rf =0.45 on alumina Rf =0.53; Rf =0.42.

The starting material can be prepared as follows:

(1) BOC-Cal-Leu-Gly-OEt 4.35 g. of BOC-Val-OH are dissolved in 50 ml. of tetrahydrofuran and the mixed anhydride is formed as described under 58 in Example 1, and coupled with 5.06 g. of H-Leu-Gly-OEt (Example 1, 57). The product melts at 1121l3 C. [a] =27.3 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf =0.7.

(2) BOC-Val-Leu-Gly-OH BOC-Val-Leu-Gly-OEt is hydrolysed in the same manner as described under 59 in Example 1. The product melts at 78 C. [a] =24.7 (c.=1 in dimethylformamide). In the thin-layer chromatogram on silica gel Rf: 0.71.

(3) H-Val-Leu-Gly-OH B O C-C ys-Gly-Asn-Leu-Ser-ThbCys-Val-Leu-Gly-Thr-Tyr-Thr- Gln-Asp (OtBu) Phe-Asn-Lys (BOC)Phe-His-Thr-Phe-Pro-Gln-Thr- Ala-Ile-Gly-Val-Gly-Ala-Pro-NH:

440 mg of the docosapeptideamide described under 43 in Example 1 are condensed in the same manner as described under 62 in Example 1 with I l B 0 C-0 ys-Gly-Asn-Leu-Ser-Thr-Oys-Val-Leu- Gly-0H. In the thin-layer chromatogram on silica gel Rf =0.29.

EXAMPLE 3 l' l H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Leu-Leu- Gly-Thr-Tyr-Thr- Gln-Asp- Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala- Pro-NH; (Leu -ealcitonine M) 200 mg. of

are covered with 3.5 ml. of ice-cold trifiuoracetic acid of strength; when the peptide derivative has completely dissolved, the mixture is heated to 30 C. and kept for 1 hour, and then poured into 200 ml. of ice-cold ether. The fine precipitate is suctioned olf, dried under vacuum over sodium hydroxide, dissolved in dilute acetic acid and freed from trifiuoracetate ions by filtration through a column of Merck ion exchange resin NII, weakly basic. The eluate is lyophilized and furnishes the acetate of Leu -calcitonine 65 M The starting material is accessible thus: 400 mg. of

B O C-Cys- Gly-Asn-Leu-Ser (tBu) Thr (tBu)-Cys-Leu-Leu- Gly-0H,

600 mg. of H Thr(tBu) Tyr(tBu) Thr(tBu)-Gln-Asp (OtBu) Phe Asn Cys(BOC) Phe His Thr(tBu)- Phe Pro Gln Thr(tBu) Ala Ile Gly Val Gly- Ala-PIO NH mg. of N-hydroxysuccinimide, mg. of dicyclohexylcarbodiimide and 6 ml. of dimethylformamide are stirred for 3 hours at 45 C. The mixture is 33 then poured into 300 ml. of ether and the precipitate is filtered ofi. The crude product is purified by countercurrent distribution in the system methanol+butfer+chloroform-l-carbon tetrachloride 11:3:6z7; buffer as in Example 1; K=0.7.

The decapeptide used as starting material is manufactured as described in Kamber and Rittel, Helv. Chim. Acta 51 (1968), 2061 for the corresponding Met -decapeptide: The starting material used is H-Leu-Gly-OMe which is reacted with Z-leucine N hydroxysuccinimide ester, the resulting Z-Leu-Leu-Gly-OMe is hydrogenated the H-Leu-Leu-Glu-OMe, HCl condensed with TRI-Cys- (TRI)-OH as shown under 9) inExample 1 of Kamber and Rittel, Helv. Chim. Acta 51, (1968), 2061 and the resulting tetrapeptide derivative is Worked up as described under 10) to 18) in the above-mentioned Application.

EXAMPLE 4 r--| H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Nva-Leu-Gly-Thr-Tyr-Thr-Gln Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-G1n-Thr-Ala-Ile-Gly-Val-Gly- Ala-Pro-NHz (N va -calcitonine M) 5 mg. of

are mixed with 0.2 ml. of 95% trifluoracetic acid and kept for 1 hour at 30 C. Then 5 ml. of ether are added, the precipitate is removed on a centrifuge, washed with a small quantity of ether and dried. The dotriacontapeptide amide obtained in form of the trifluoracetate can be converted into the acetate by ion exchange.

The starting material can be prepared thus:

( 1) Z-Nva-Leu-Gly-OMe 4.3 Grams of Z-L-Nva-2,4,S-trichlorophenyl ester (J. chem. Soc. (c) 1968, 715) and 2.2 g. of H-Leu-Gly-OMe are mixed with ml. of dimethylformamide and the reaction mixture is stirred for 48 hours at 35 C., then diluted with much chloroform, cooled in ice and agitated three times with 5% ice-cold potassium carbonate solution and three times with ice water. The chloroform solution is dried with sodium sulphate and then evaporated to dryness, finally under 0.1 mm. Hg to remove any residues of dimethylformamide. The residue is dissolved in chloroform and chromatographed on a column, prepared in chloroform, of silica gel, using for the elution a linear graduated chloroform-chloroform+methanol(1:1) (1 litre each). Fractions of 100 ml. each are collected, evaporated to dryness and the purity of the residue is inspected by thin-layer chromatography on silica gel plates and the pure fractions are combined. In toluene+acetone (1:1) the product reveals an R) value of 0.4.

A solution of 1.7 g. of the above tripeptide in methanol is hydrogenated While being vigorously stirred in the presence of 400 mg. of palladium carbon (10% Pd). When hydrogen is no longer being absorbed, the catalyst is filtered off and the filtrate evaporated to dryness. The resulting colourless resin can be used for the further reaction as it is obtained.

(3 TRI-Cys- (TRI) Nva-Leu-Gly-OMe 2.5 Grams of TRI-Cys(TRI)-OH and 1.1 g. of the above H-Nva-Leu-Gly-OMe are mixed with 40 ml. of acetonitrile and then 1.3 g. of dicyclohexylcarbodiimide are stirred in. The whole is stirred for 20 hours at 27 C., then the precipitate formed is suctioned off and extracted with 1 litre of chloroform. The eluate is evaporated to dryness and the residue triturated with petroleum ether. The petroleum ether solution is decanted and the insoluble product is dried and purified by 3 precipitations from petroleum ether. The tetrapeptide derivative is obtained as an amorphous, solid powder which reveals in thin-layer 34 chromatography on silica gel plates in chloroform-I-methanol (98:2) an R value of 0.62.

(4) H-CysUDRI)Nva-Leu-Gly-OMe 980 mg. of TRI-Cys(TRI)Nva-Leu-Gly-OMe are dissolved at 30 C. in 25 ml. of 75% acetic acid and then kept for 1 hour, then evaporated to dryness and the residue is repeatedly triturated with ether. The ether-insoluble acetate of H-Cys (TRI)-Nva-Leu-Gly-OMe is dried at 35 C. under vacuum and further worked up as it is.

(5) DPC-Ser(tBu) Thr tBu) Cys- (T RI) Nva-Leu-Gly-OMe A solution of 750 mg. of DPC-Ser(tBu)-Thr(tBu)- NHNH in 6 ml. of dimethylformamide is cooled to 20 C. and 3.5 ml. of N-hydrogen chloride in ethyl acetate are added. Then 0.16 ml. of tertiary butylnitrite is added and the reaction mixture is kept for 15 minutes at -10" C.; then a solution, cooled at 10 C., of 500 mg. of H-Cys(TRI)Nva-Leu-Gly-OMe in 4 ml. of dimethylformamide and 0.7 ml. of triethylamine are added and the batch is kept for 30 minutes at 10" C. and then for 18 hours at 0 C., then evaporated under a high vacuum at 40 C. bath temperature to a volume of about 5 ml., and the product is precipitated with ice water. It is thoroughly triturated, then suctioned off, washed with ice water and the crude hexapeptide derivative is dried in a desiccator over calcium chloride, and purified by three reprecipitations from ethyl acetate+cyclohexane. In the thin-layer chromatography on silica gel plates in toluene+acetone (7:3) the product reveals an Rf value of 0.47.

(6) H-Ser (tBu) Thr (tB u) Cys (TRI Nva-Leu-Gly-OH A solution of 430 mg. of DPC-Ser(tBu)-Thr(tBu)-Cys- (TRI)-Nva-Leu-Gly-O-Me in 10 ml. of acetic acid is kept for 7 hours at 28 C., then evaporated to dryness in a rotary evaporator at 35 C. bath temperature, dried for 3 hours at 35 C. under 001 mm. Hg pressure, then dissolved in 30 ml. of methanol, mixed with 3 ml. of N-sodium hydroxide solution, stirred for 1 hour at 28 C., 2 ml. of glacial acetic acid are added and the whole is evaporated to dryness in a rotary evaporator at 35 C. bath temperature. The residue is triturated with ice Water while adjusting the pH value to 6-7, suctioned off, rinsed with ice water and dried over calcium chloride in desiccator.

(7 BOC-Cys- (TRI) Gly-Asn-Leu-Ser (tBu) Thr (tBu) Cys (TRI Nva-Leu-GlyOI-I A mixture of 270 mg. of BOC-Cys('IRI)-Gly-Asn- Leu-NH-NH and 4 ml. of dimethylformamide is cooled to 20 C. and 1.2 ml. of N-hydrogen chloride in ethyl acetate are added. Then 0.044 ml. of tertiary butylnitrite is added and the whole is kept for 15 minutes at --10 C. Then a solution, cooled at -10 C., of 230 mg. of H-Ser(tBu)-Thr(tBu)-Cys(TRI)-N va-LeuGly-OH in 4 ml. of dimethylformamide, 1 ml. of water and 0.25 ml. of triethylamine is added. The batch is kept for 18 hours at 0 C., then evaporated to a small volume under 0.01 mm. Hg at 35 C. bath temperature, and the decapeptide is precipitated with water. The precipitate is triturated, suctioned off and dried over calcium chloride in a desiccator. The powder is suspended in 7 ml. of methanol at 0 C., thoroughly triturated and then suctioned 01f.

mg. of the decapeptide obtained under (7) are dis solved in 20 ml. of dimethylformamide and within 1 hour this solution is vigorously dropped in a solution of mg. of iodine in 100 ml. of methanol. The batch is stirred for 1 hour longer, then decolorized at 0 C. by adding N-sodium thiosulphate solution, then concentrated to a volume of about 20 ml. in a rotary evaporator and precipitated with 300 ml. of ether. The product is suctioned off, dried at 30 C. under vacuum and the powder is then triturated with water, suctioned off and dried over calcium chloride in a desiccator.

(9) Z-Asp (OtBu) -Phe-OCH 30.3 Grams of Z-Asp(OtBu)-ONP and 18.3 g. of H- Phe-OCH .HCl are together dissolved in 150 ml. of dimethylformamide, and 11.8 ml. of triethylamine are dropped into the clear solution. The resulting suspension is stirred for 20 hours at room temperature, during which it turns deep yellow, then it is concentrated to about 100 ml. under vacuum and in 1 litre of ethyl acetate +chloroform (4:1) agitated three times with 5% citric acid, 19 times with about 2 N-sodium carbonate solution and with saturated sodium chloride solution until a neutral reaction has been reached. The crude product forms a yellow oil which is treated with active carbon in ether and, after having been seeded, crystallizes from 650 ml. of ether-l-hexane (1: 1) in a refrigerator. It forms colourless needles melting at 74.5 to 76.5 C. In the thin-layer chromatogram on silica gel in the system chloroform +rnethanol (95:5) Rf=0.74, in the system chloroform +acetone (75:25) Rf=0.65.

(l) H-Asp(tB11)-Phe-OCH 48.6 Grams of Z-Asp(OtBu)-Phe-OCH in 700 ml. of methanol are mixed with 33.5 ml. of 3 N-hydrogen chlo ride in dioxan and 5 g. of palladium carbon catalyst are decarbobenzoxylated at room temperature in a duckshaped vessel. On cessation of the absorption of hydrogen the lwhole is filtered and the filtrate evaporated, to yield 38.7 g. of a white foam. In the thin-layer chromatogram on silica gel in chloroform-l-methanol (9:1) Rf=0.60, in chloroform-l-acetone (1:1) Rf==0.58,

The product is used as it is obtained in the following condensation.

A clear, pale-yellowish solution is prepared of 38.6 g. of the resulting H-Asp(OtBu)-Phe-OCH .CHl and 42.0 g. of Z-Gln-ONP in 170 ml. of dimethylformamide, and 13.9 ml. of triethylamine are slowly stirred in. The resulting orange suspension is stirred for 24 hours at -35 C. bath temperature. During this time another ml. of dimethylformamide are added as well as 1.39 ml. of triethylamine.

For working up the batch it is dissolved in 4 litres of chloroform and washed in a 20-stage counter-current distribution apparatus (phase volume: bottom phase 400 ml., upper phase 200 ml. per vessel) successively with 1 litre of 5% citric acid solution, 400 ml. of saturated sodium chloride solution, 6 litres of about 2 N-sodium carbonate solution and 2.8 litres of saturated sodium chloride solution. After drying and evaporating the tripeptide derivative crystallizes from 1.8 litres of ethanol in a refrigerator slowly, to yield Z-Gln-Asp(OtBu)-Phe-OCH melting at l86188 C.

In thin-layer chromatogram silica gel the following Rf values are obtained:

In chlorofom-I-methanol (9:1) Rf=0.39; in chloroform+acetone (1:1) Rf=0.24; Rf =0.69, Rf =0.46, Rf =0.65, [a] =28:1 (c.=1.3% in dimethylformamide).

12 H-Gln-Asp(OtBu)-Phe-OCH A solution of 7.55 g. of Z-Gln-Asp(OtBu)-Phe-OCH in 400 ml of methanol is mixed with 4.1 ml. of 3 N- hydrogen chloride in dioxan and hydrogenated in the presence of 2 g. of palladium carbon (10% Pd), then the catalyst is suctioned off and the filtrate evaporated to yield H-Gln-Asp(OtBu)-Phe-OCH .HCL in form of a colourless foam. In the thin-layer chromatogram on silicagel the following R) values are obtained: In chloroform-{mefollowing Rf values are obtained: In chloroform-l-methanol (9:1) Rf=0.13; in chloroform+acetone (25:75)

(13) Z-Thr(tBu)-Gln-Asp( OtBu -Phe-OCH The whole quantity of the hydrochloride of (12) to gether with 7.4 g. of Z-Thr(tBu)-OSU is dissolved at room temperature in 14 ml. of dimethylformamide, and 1.72 ml. of triethylamine are dropped in while cooling in an ice bath. The brownish suspension is then stirred for 20 hours at room temperature, worked up in the usual manner in much ethyl acetate (washing 3 times each with 5% citric acid and about 2 N-sodium carbonate solution, washing neutral with saturated sodium chloride solution, drying over sodium sulphate and evaporation at 3040 C. under vacuum) the crude product is treated with active carbon in ethanol and recrystallized in a refrigerator from ml. of ethanol; it melts at 155161 C.

In the thin-layer chromatogram on silica gel the following Rf values are found: In chloroform+methanol (9:1) Rf=0.52; in cyclohexane-l-acetone (3:7) Rf=0.48; Rf =0.48; Rf =0.76. [u] =4:O.5 (c.=2.3% in dimethylformamide).

( 14) H-Thr(tBu)-Gln-Asp(OtBu)-Phe-OCH 478 mg. of Z-Thr(tBu)-Gln-Asp(OtBu)-Phe-OCH in 150 ml. of methanol are hydrogenated neutral at room temperature with 100 mg. of palladium carbon (10% Pd), to yield 395 mg. of a colourless foam of H-Thr(tBu)- Gln-Asp(OtBu)-Phe-OCH which is used in the following condensation without previous purification.

In the thin-layer chromatogram on silica gel the following Rf values are found: In chloroform-i-methanol (1:1) Rf=0.75; in chloroform+methanol (9:1) Rf: 0.17; in acetone Rf=0.18; Rf =0.23; Rf =0.l2.

687 mg. of Z-Tyr(tBu)OH-dicyclohexylammonium salt in chloroform are agitated with aqueous citric acid and the resulting free acid (a clear oil) in 6.5 ml. of tetrahydrofuran is mixed with 0.139 ml. of N-methylporpholine. At 22 C. 0.170 ml. of chloroformic acid isobutyl ester is added and the whole is stirred for half an hour at 22 to 10 C. Then a precooled solution of the above-described H-Thr(tBu)-Gln-Asp(OtBu)-Phe- OCH in 15 ml. of tetrahydrofuran is dropped in, rinsing being performed with 5 ml. of the same solvent. After half an hour at 10 C. the batch is stirred on for 15 hours at room temperature, then concentrated under vacuum and worked up in the usual manner in ethyl acetate (see under (13) The crude product is dissolved in 15 ml. of ethyl acetate, precipitated with 40 ml. of ether and then crystallized from methanol in a refrigerator; it forms short, thick needles which on drying under a high vacuum at 50 C. efiloresce. Melting point: 1691730 C.

In the thin-layer chromatogram on silica gel the following Rf values are found: In chloroform-i-methanol (9:1) Rf=0.46; in chl0rof0rm+methan0l (1:1) Rf=0.95; in chloroform acetone (1:1) R 0.44; Rfgg 0.61; Rf 068; Rf =0.73. [a] =-54i0.5 (c.= 2.0% in dimethylformamide).

2.36 Grams of Z-Tyr(tBu)-Thr(tBu)-Gln-Asp(OtBu)- Phe-OCH in 450 ml. of methanol are hydrogenated in the usual manner with 500 mg. of palladium carbon (10% Pd) at room temperature, to form a colourless foam which is unitary according to its thin-layer chromatogram and is further used as it is obtained.

In the thin-layer chromatogram on silica gel the following Rf values are found: In chloroform-{methanol (:5) Rf=0.22; Rf =0.42.

The product of (16) together with 1.48 g. of Z-Thr 7 5 (tBu)-OSU is dissolved in 3 ml. of dimethylformamide and 37 stirred for 21 hours at room temperature. The reaction solution is diluted with much ethyl acetate and worked up as usual (see under (13)). The crude product is dissolved with heating in 30 ml. of ethyl acetate-l-methanol (9: 1), then cooled in an ice bath and precipitated with 80 ml. of ether. The resulting product is a colourless, amorphous powder melting at l46l48 C.

In the thin-layer chromatogram on silica gel the following Rf values are obtained: In chloroform-l-methanl (9: 1) Rf=0.55; in chloroform+acetone (1:1) Rf-=0.60; RM: 0.43. [u] =|-6i0.5 (c.=2.0 in dimethylformamide).

(18) Z-Thr (tBu -Tyr (tBu) -Thr (tBu -Gln-Asp OtBu) Phe-NH-NH A solution of 1.91 g. of Z-Thr(tBu)-Tyr(tBu)- Thr(tBu)-Gln-Asp (OtBu)-Phe-OCH in 80 ml. of methanol is mixed with 8 ml. of hydrazine hydrate, then kept for 22 hours at room temperature and the product which has settled out is filtered 011 and dried under a high vacuum at 60 C., to yield the microcrystalline hydrazide melting at 226229 C. with decomposition.

In the thin-layer chromatogram on silica gel the following Rf values are found: In chloroform-l-methanol (9:1) Rf=0.32; in cyclohexane-i-acetone (3:7) Rf=0.23; Rf =0.34. [a] =+4i1 (c.=1.0 in dimethylformamide).

(19) Z-Lys (BOC) -Phe-OMe 6.9 ml. of triethylamine are stirred at room temperature into 25.0 g. of Z-Lys(BOC)-ONP and 10.7 g. of H-Phe- OMe, HCL in 70 ml. of dimethylformamide and the mixture is stirred on for 18 hours, then diluted with ethyl acetate and washed with potassium carbonate solution until it is free from nitrophenol, agitated with 0.1 molar citric acid and water, dried over sodium sulphate and evaporated to dryness under vacuum. From ethyl acetate+hexane the protected, crystalline dipeptide melting at 78-80 C. is obtained.

In the thin-layer chromatogram on silica gel in the system chloroform-Facetone (8:2) Rf=0.45.

A solution of 27 g. of the above dipeptide methyl ester in 135 ml. of warm methanol is mixed at room temperature with 25 ml. of hydrazine hydrate and left to itself for 16 hours. The crystallizate is mixed with 135 ml. of water, suctioned off and thoroughly washed with water; after recrystallization from aqueous methanol it melts at 173- 174 C. In the thin-layer chromatogram on silica gel in the system chloroform-l-methanol (95 :5) Rf=0 21) Z-Lys (*BOC) -Phe-His-OMe 5.4 Grams of Z-Lys(BOC)-Phe-NH-NH in 40 ml. of dimethylformamide are mixed at 16 C. with 6.8 ml. of 3.66 N-hydrochloric acid in dioxan and then with 1.5 ml. of tertiary butylnitrite. After minutes at 10 C. to C. 3.5 ml. of triethylamine are added. Then 3.64 g. of solid H-His-OMe, 2HC1 are added and then 4.2 ml. of triethylamine are dropped in. The batch is allowed to warm up to 0 C. within 1 hour, during which a pH value of about 7 is adjusted by adding a total of 0.8 ml. of triethylamine. The whole is stirred overnight at 0 C., then poured into 250 ml. of water and the smeary product is obtained in pulverulent form by trituration with water. In the thin-layer chromatogram on silica gel in chloroform+methanol (9:1) Rf=0.4.

(22) H-Lys (BOC) -Phe-His-OMe 6.8 Grams of Z-Lys(BOC)-Phe-His-OMe in 140 ml. of methanol are hydrogenated in the presence of 1 g. of 10% palladium carbon. On completion of the hydrogenation the catalyst is filtered off, the filtrate evaporated to dryness and the residue immediately further worked up.

38 2s Z-Asn-Lys(BOC)-Phe-His-OMe 5.4 Grams of H-Lys('BOC)-Phe-His-OMe and 4.5 g. of Z-Asn-ONP in 20 ml. of dimethylformamide are stirred for 20 hours at room temperature. The peptide derivative is then precipitated with ethyl acetate, filtered off, washed with ether and recrystallized from methanol whereupon the product melts at 182 to 183 C. In the thin-layer chromatogram on silica gel Rf =0.57. [a] =-28 (c.:1 in dimethylformamide).

( 24) Z-Asn-Lys (BOC) -Phe-HisNH-NH 3.97 Grams of Z-Asn-Lys(BOC)-Phe-His-OMe are dissolved in 8 ml. of warm dimethylformamide and 12 ml. of methanol. While this solution still has a temperature of about 30 C., it is mixed with 2.5 ml. of hydrazine hydrate and kept for 20 hours at room temperature. The peptide hydrazide is precipitated with water, then filtered off, washed free from hydrazine with water and recrystallized from ethanol; it melts at ZOO-201 C. In the thin-layer chromatogram on silica gel Rf =0.5.

(25) H-Phe-Pro-OH Z-Phe-Pro-OH is converted into the free dipeptide by catalytic reduction with palladium carbon in methanol-1- water (4:1). The dipeptide is isolated by concentrating the hydrogenating solution to a small volume, after having filtered off the catalyst, and addition of acetone, in fact as the crystalline dipeptide monohydrate melting at 125128 C.

(26) Z-Thr (tBu) -Phe-Pro-OH 20.2 Grams of Z-Thr(tBu)-OSU, 13.3 g. of H-Phe-Pro- OH (monohydrate) and 6.54 ml. of trlethylamine are dissolved in ml. of dimethylformamide, kept overnight at about 20 C. and then concentrated under a high vacuum until a sticky substance has formed which is dissolved in 500 ml. of ethyl acetate and washed with 5 X 100 ml. of 5% tartaric acid solution and then with water until the washings run neutral. The organic phase is evaporated to dryness and the residual, solid foam is powdered and dried under a high vacuum at 40 C. Two more precipitations from ethyl acetate+petroleum ether furnish 13.3 g. of amorphous, chromatographically pure tripeptide derivative having an unsharp melting range of about 75 to C. In the thin-layer chromatogram on silica gel Rf =0.68; Rf =0.57.

(27) Z-Ile-Gly-OMe A suspension of 2.23 g. of Z-Ile-OH-dicyclohexylammonium salt in ethyl acetate is acidified with 0.2 molar citric acid. The resulting ethyl acetate solution is washed neutral, dried and evaporated to dryness. The residue is dissolved in 15 ml. of acetonitrile, and the solution is mixed with 750 mg. of H-Gly-OMe, HCl and, while stirring at 0 C., with 0.84 ml. of triethylamine are added. After 10 minutes 1.24 g. of dicyclohexyl carbodiimide are added and the whole is stirred overnight at 0 C. The precipitate is filtered off and the filtrate evaporated to dryness, and the residue is taken up in 30 ml. of ethyl acetate and filtered. The ethyl acetate solution is washed with 0.2 molar citric acid solution and saturated sodium bicarbonate solution, dried and concentrated to about 10 ml. under vacuum. On addition of 25 ml. of hexane the protected dipeptide, melting at 120-l22 C., crystallizes out. Rf=0.53 in the system chloroform+methanol (:5) in thin-layer chromatography on silica gel.

(28) H-Ile-Gly-OMe A solution of 3.36 g. of Z-Ile-Gly-OMe in ml. of methanol and 10 ml. of N-hydrochloric acid is hydrogenated in the presence of 0.5 g. of 10% palladium carbon. The catalyst is filtered oif and the solvent completely evaporated. The resulting foam is unitary in the thin-layer chromatogram on silica gel; Rf=0.26 in chloroform-[- methanol (95 :5).

39 (29 Z-Ala-Ile-Gly-OMe While stirring 2.39 g. of the above dipeptide ester hydrochloride and 3.78 g. of Z-Ala-ONP in 40 ml. of dimethylformamide, 1.4 ml. of triethylamine are added and the resulting suspension is stirred overnight at room temperature, diluted with ethyl acetate and washed free from nitrophenol with dilute potassium carbonate solution, and then further washed with 0.1 molar citric acid and water. During the agitation part of the tripeptidc derivative remains undissolved and is filtered off. The ethyl acetate solution is dried and evaporated to dryness. The residue likewise consists of pure product melting at 190-191 C. In the thin-layer chromatogram on silica gel in the system chloroform-l-methanol (95:5) Rf=0.5.

(3 H-Ala-Ile-Gly-O Me 2.0 Grams of Z-Ala-Ile-Gly-OMe are dissolved with gentle heating in 40 ml. of methanol and then hydrogenated in the presence of 300 mg. of palladium carbon Pd). On completion of the hydrogenation the catalyst is filtered off and the filtrate evaporated completely to dryness. The residue is found to be unitary in the thinlayer chromatogram and is immediately further worked up.

(31) Z-Thr (tBu -Ala-Ile-Gly-OMe 1.36 Grams of H-Ala-Ile-Gly-OMe and 2.5 g. of Z- Thr(tBu)-OSU in 3 ml. of dimethylformamide are stirred for hours at room temperature. The tetrapeptide derivative is precipitated with ether, filtered off and washed with ether; after recrystallization from ethanol it melts at 229230 C. [a] =--43 (c.=1 in methanol. Rf=0.55 in the system chloroform+methanol (95:5) on silica gel.

(32) H-Thr(tBu)-Ala-Ile-Gly-OMe A solution of 5.66 g. of the above carbobenzoxy compound in 400 ml. of warm methanol is hydrogenated in the presence of 1 g. of palladium carbon (10% Pd). The catalyst is filtered off and the methanol expelled under vacuum at 40 C. The solid residue is immediately further worked up. Rf=0.2 in the system chloroform-i-methanol (95:5) on silica gel.

(3 3) Z-Gln-Thr tBu) -Ala-Ile-Gly-OMe 4.6 Grams of the H-Thr(tBu)-Ala-Ile-Gly-OMe described under (32) in ml. of dimethylformamide are mixed with 3.5 g. of Z-Gln-ONP and stirred at room temperature until the mixture has turned solid. It is then kept overnight and diluted with ether, the precipitate is filtered off and washed with ether until it is free from nitrophenol. The protected pentapeptide reveals in the thin-layer chromatogram on silica gel an R value of 0.14 in the system chloroform+methanol (95 :5). It melts above 250 C.

(34) H-Gln-Thr (tBu) -Ala-Ile-Gly-OMe,HCl

14.4 Grams of the pentapeptide derivative described under (33) are suspended in 800 ml. of 80% methanol and heated for some time at 50 C. The suspension is cooled to room temperature, mixed with 20.8 ml. of hydrochloric acid and 3 g. of palladium carbon (10% Pd) and hydrogenated until the absorption of hydrogen ceases and the starting material has dissolved. The catalyst is filtered off and the filtrate evaporated at 40 C. under vacuum, and the residue is dehydrated by being twice evaporated under a high vacuum with dimethylformamide. The residue is used as it is obtained. Rf =O33 in the thin-layer chromatogram on silicagel.

(35) Z-Thr(tBu)-Phe-Pro-Gln- Thr(tBu)-Ala-Ile-Gly-OMe 12.0 Grams of the pentapeptide-methyl ester hydrochloride described under (34) are dissolved in 80 ml. of dimethylformamide and 13.3 g. of Z-Thr(tBu)-Phe-Pro- OH and then 5.75 g. of N-hydroxysuccinimide are stirred in at room temperature, and then at 0 C. 2.76 ml. of triethylamine and 6.2 g. of dicyclohexylcarbodiimide. The whole is stirred at 0 C. until it turns thick and then kept overnight at 0 C., concentrated to about 50 ml. under a high vacuum and the product is precipitated with 300 ml. of ether. The isolated material is washed with 0.05-molar citric acid and water and dried under a high vacuum at 40 C. and purified by recrystallization from about 1 litre of isopropanol, to yield 18.2 g. of the protected octapeptide derivative. Rf =0.27 in the thinlayer chromatogram on silica gel.

(36) Z-Thr(tBu)-Phe-Pro-Gln-Thr(tBu)-Ala-Ile-Gly-OH 10.9 Grams of the octapeptide methyl ester described under (35) are dissolved with heating at 70 C. in 190 ml. of 90% methanol; the solution is cooled to room temperature and 30 ml. of N-sodium hydroxide solution and, 10 minutes later, 160 ml. of water are added in small portions. The whole is then filtered clear and from the filtrate the product is precipitated by pouring in 600 ml. of 0.05 N-ice-cold hydrochloric acid. The precipitate is filtered off and washed neutral with water. The product is unitary in the thin-layer chromatogram on silica gel methanol.

(Rf =0.45) and can be recrystallized from aqueous methanol.

(37) H-Thr(tBu)-Phe-Pro-Gln-Thr (tBu -Ala-Ile-Gly-OH A solution of 3.6 g. of the Z-Thr(tBu)-Phe-Pro-Gln- Thr(tBu)-Ala-Ile-Gly-OH obtained in (36) in 100 ml. of acetic acid of strength is hydrogenated in the presence of 0.5 g. of palladized carbon (10% Pd). On cessation of the hydrogen absorption the catalyst is suctioned off and the solution evaporated to dryness. The acetate of the octapeptide derivative, obtained in the form of a granular snow, is dried under a high vacuum. Rf =0.2l in the thin-layer chromatogram on silica gel.

(3 8) Z-Asn-Lys (BOC -Phe-His-Thr (tBu Phe-Pro-Gln-Thr (tBu -Ala-Ile-Gly-OH A solution of 11.25 g. of Z-Asn-Lys(BOC)-Phe-His hydrazide in 65 ml. of dimethylformamide is mixed within 2 minutes at 20 to --25 C. with 8.4 ml. of 4.2 N- hydrogen chloride in dioxane. Then 2.1 ml. of tertiary butylnitrite are added at 15 to -20 C. and the whole is kept for 15 minutes at 15 C., cooled to 20 C. and 4.8 ml. of triethylamine are added and then a solution of 9.0 g. of the product described in (37) in 210 ml. of dimethylformamide of strength, while maintaining the internal temperature at l5 C. by vigorous cooling. Within 1 hour the batch is warmed to 0 C., while main-' taining the pH value at 7 to 8 by occasional addition of triethylamine (total of triethylamine added: 3.5 ml.). The batch is stirred overnight at 0 C., then poured into 3 litres of ether, the floccular precipitate is filtered off and washed twice with ether and once with water. The crude product is dissolved in 500 ml. of warm methanol and reprecipitated by being poured into 1.5 litres of 1% acetic acid. The product is filtered off, twice washed with Water, and once more precipitated as described. Rf =033 (on silica gel plates).

(3 9) H-Asn-Lys( BOC) -Phe-His-Thr(tBu) Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-OH A solution of 1.7 g. of the above protected dodecapeptide in 100 ml. of 80% acetic acid is hydrogenated in the usual manner in the presence of 0.5 g. of palladized carbon (10% Pd). The catalyst is filtered oil and the filtrate considerably concentrated in a high vacuum at 30 C., and the residue is lyophilized with tertiary butanol. The product is obtained in a quantitative yield; it 

1. PEPTIDES OF THE FORMULA I H-CYS-GLY-ASN-LEU-SER-THR-CYS-MET-LEU-GLY-THR-TYR-THR-GLNASP-PHE-ASN-LYS-PHE-HIS-THR-PHE-PRO-GLN-THR-ALA-ILEGLY-VAL-GLY-ALA-PRO-NH2, WHERE THE CYS RESIDUES ARE JOINED, IN WHICH THE AMINOACID IN POSITION 8 IS REPLACED BY VALINE, NORVALINE, LEUCINE, ISOLEUCINE, NORLEUCINE OR A-AMINOBYTYRIC ACID, AND THOSE WHICH CONTAIN GLYCINE IN POSITION 12 AND THOSE WHICH CONTAIN METHIONINE IN POSITION 8 AND GLYCINE IN POSITIONS 12 AND 18, OR AT LEAST IN POSITION 12, THEIR ANTIPARALLEL DIMER AND NA-ACYL DERIVATIVES WHEREIN ACRYL IS LOWER MONOBASIC OR DIBASIC ALKANOYL, LOWER ALKYLOXYCARBONYL, BENZYLOXYCARBONYL, L-PYROGLUTAMYL, CARBAMOYL, N-LOWER ALKYLCARBAMOYL, N-PHENYLCARBAMOYL, OR N-PHENYLTHIOCARBAMOYL, OR DESAMINO1-DERIVATIVES OF THE MONOMERIC OR DIMERIC PEPTIDES, AS WELL AS THERAPEUTICALLY ACCEPTABLE ACID ADDITIONS SALTS AND COMPLEXES WITH A MEMBER SELECTED FROM THE GROUP CONSISTING OF ZINC PHOSPHATE, ZINC PYROPHOSPHATE, ZINC HYDROXIDE, GELATINE, POLYPHLORETIN PHOSPHATE AND POLYGLUTAMIC ACID, OF THE SAID MONOMERIC OR DIMERIC PEPTIDES WITH THE PROVISO THAT ALL AMINOACID RESIDURS OTHER THAN GLYCINE ARE TO THE L-CONFIGURATION. 